在 MIT 7.05 生物化学课堂上，当著名癌症代谢学家 Matthew Vander Heiden 教授结束蛋白质与酶学板块、正式开启糖类化学与新陈代谢大幕时，他在黑板上向全班抛出了三个看似平常却直击生命底层逻辑的灵魂追问：

第一，六碳醛糖（己醛糖）在有机化学上共有 2⁴ = 16 种旋光异构体，为什么地球上几乎所有生命都不约而同地选中了 D-葡萄糖作为血液循环糖与核心能量货币前体，而不是甘露糖或半乳糖？第二，土豆（淀粉）和木头（纤维素）都是由完全相同的 D-葡萄糖单体聚合而成的同多糖，含有完全相同的卡路里，为什么我们每天吃土豆充饥、用木头盖房子，而从来没有人用土豆盖房子或啃木头当早餐？第三，如果高中生物告诉我们“ATP 是细胞的直接能量通货”，那为什么超市货架上从来没人卖“纯 ATP 早餐麦片”？为什么一旦切断氧气与营养供应（如心肌梗死或氰化物中毒），人体细胞内的 ATP 储备只够支撑不到几秒钟就会彻底崩溃？

要回答这三个问题，我们不能靠死记硬背化学结构式或代谢途径图，而必须握住生物化学的两把金钥匙：立体化学构象（Stereochemistry & Conformation）与生物能学热力学（Bioenergetics & Thermodynamics）。从单糖分子内的半缩醛成环如何锁住活泼醛基，到 α/β 糖苷键如何决定多糖是盘曲成储能螺旋还是伸展成刚性微纤维；从胞内糖原为何用无机磷酸（P_i）进行“加磷酸解”而非像消化道那样“水解”，到细胞如何利用吉布斯自由能方程 Δ G = Δ G'^° + RTln([产物]/[反应物]) 和高能磷酸键、高能硫酯键去对抗宇宙熵增——本章将带你边听 MIT 7.05 课堂推演，边把王镜岩《生物化学》第 4 版第 9、15、16 章与华农考研（338/802）近二十年真题彻底拆透。

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1.1单糖的立体化学、环化半缩醛与变旋现象：为什么自然界偏偏选中 D-葡萄糖作血糖？

一、糖类的化学本质与四大生物学功能

在日常英语中，糖类被称为 Carbohydrate（碳水化合物）。Vander Heiden 教授在 L12 开篇便在黑板上写下了这个词的词源拆解：“Carbo-（碳）”加上“Hydrate（水合物）”，因为绝大多数基础糖类都符合通式 C_n(H_2O)_n。但他紧接着提醒大家：千万不要用“氢氧原子比 2:1”来定义糖类！在生物界中，许多单糖衍生物会引入氮、磷、硫等杂原子，或者脱去氧原子。

按照王镜岩《生物化学》第 4 版的严格标准，糖类（Carbohydrates，又称糖质）：是多羟醛、多羟酮或其衍生物，或水解时能产生这些化合物的多聚体 2017·判断。为什么不能用经验式 (CH_2O)_n 来定义？注意：(CH_2O)_n 只是经验式，鼠李糖（C_6H12O₅）和 2-脱氧核糖（C_5H10O₄）是真真正正的糖类，但它们的 H:O 并非 2:1；反之，甲醛（CH_2O）、乙酸（C_2H4O2）和乳酸（C_3H6O3）虽然符合 (CH_2O)_n，却根本不是糖类 2017·判断！

根据聚合度的不同，糖类可分为四大类：单糖（Monosaccharides）（不能被水解成更小分子的糖）、寡糖（Oligosaccharides）（由 2～ 10 个单糖通过糖苷键连接而成，以二糖最为常见）、多糖（Polysaccharides）（水解产生 10 个以上单糖分子的高聚物，分同多糖和杂多糖；寡糖与多糖之间并无绝对界线，二者常合称聚糖 Glycan），以及糖与蛋白质或脂质共价结合形成的糖缀合物（Glycoconjugates，又称结合糖或复合糖）。在生命体中，糖类的四大生物学功能是：① 作为主要能源物质（如葡萄糖、淀粉、糖原）；② 作为生物体的结构成分（如纤维素、壳多糖、肽聚糖）；③ 在体内转变为其他物质，为合成氨基酸、核苷酸、脂肪酸等提供碳骨架；④ 作为细胞识别与信号转导的生物信息分子（如糖蛋白、糖脂表面的寡糖链） 2008·填空 2019/2021·简答。

二、从三碳糖出发：手性碳、Fischer 投影式、D/L 构型与差向异构体

生物体中最简单的单糖有 3 个碳原子，称为丙糖（Trioses），分子式为 C₃(H_2O)₃。Vander Heiden 教授在黑板上画出了满足这一分子式的两种同分异构体（Isomers）：

1. 把羰基放在碳链末端（C1），得到一个醛基（-CHO），这种单糖叫甘油醛（Glyceraldehyde），属于醛糖（Aldose）；
2. 把羰基放在碳链中间（C2），得到一个酮基（C=O），这种单糖叫二羟丙酮（Dihydroxyacetone），属于酮糖（Ketose）。

仔细观察这两个三碳糖的 C2 碳原子：二羟丙酮的 C2 是羰基碳，C1 和 C3 都是 -CH_2OH，因此 二羟丙酮是单糖（丙糖至庚糖）中唯一不含手性碳原子（C^*）、没有旋光性（无光学活性）的糖 ⚑华农高频（正如甘氨酸是 20 种标准氨基酸中唯一无手性碳的氨基酸一样！）。相反，甘油醛的 C2 连有 4 个不同的基团（-H、-OH、-CHO、-CH_2OH），是具有光学活性的最简单单糖，被选作确定所有单糖 D/L 构型的参照标准。

在纸面上表示手性单糖立体结构时，化学家采用 Fischer 投影式（Fischer projection）：碳链竖直排列，将氧化态最高（编号最小）的羰基碳放在最上方；水平方向化学键朝向纸面外（朝向读者），竖直方向化学键指向纸面后（远离读者）。注意：Fischer 投影式只能在纸面内旋转 180^°（构型不变）；若在纸面内旋转 90^° 或翻转出纸面，就会变成它的镜像对映体！

当碳链延长到四碳（丁糖，如 D-赤藓糖 D-erythrose 与 D-苏阿糖 D-threose）、五碳（戊糖）、六碳（己糖）时，分子中会出现多个手性碳原子（C^*）。此时如何判定整个单糖分子的立体异构关系？请牢记以下四大黄金法则：

1. 旋光异构体数目公式：含有 n 个手性碳原子（C^）的单糖，有 2ⁿ 个旋光异构体，组成 2^n-1 对对映体；己醛糖含 4 个 C^（C2、C3、C4、C5），共有 2⁴ = 16 个旋光异构体（8 对对映体）；己酮糖含 3 个 C^*（C3、C4、C5），共有 2³ = 8 个旋光异构体（4 对对映体）。
2. 酮糖羰基的位置规律：Vander Heiden 教授在课堂上特别点破了一个生化底层规律——为什么生物界中无论是五碳酮糖（核酮糖、木酮糖）还是六碳酮糖（果糖），它们的酮基永远在 C2 位？因为细胞在代谢中需要用异构酶（Isomerase）经“烯二醇中间物（Enediol intermediate）”在醛糖（C1 羰基）和酮糖之间相互转化，只有当酮基紧挨着 C1（即位于 C2 位）时，才能通过一步烯二醇互变与醛糖自由切换！
3. D/L 构型的判定标准：当分子中有多个手性碳时，单糖的 D/L 构型仅由编号最大、即离羰基碳最远的那一个手性碳原子（戊糖看 C4，己糖看 C5）的构型决定：在 Fischer 投影式中，离羰基最远 C^* 的羟基（-OH）朝右（与 D-甘油醛相同）即为 D 型，朝左即为 L 型。自然界中的单糖绝大多数是 D-型糖（与蛋白质中绝大多数是 L-型氨基酸恰成鲜明对比），但也存在少数重要的 L-型单糖，如 阿拉伯胶中的 L-阿拉伯糖、细菌细胞壁和植物糖苷中的 L-鼠李糖（6-脱氧-L-甘露糖）以及 ABO 血型抗原中的 L-岩藻糖（6-脱氧-L-半乳糖）。

• 千万注意：单糖的 D/L 构型是人为选定参照物的相对构型，与该糖实际的旋光方向（右旋 + 或左旋 -）毫无直接关系！例如 D-葡萄糖是右旋的（+52.6^°，故称右旋糖 Dextrose），但自然界最丰富的酮糖 D-果糖 虽然是纯正的 D 型构型，其比旋光度却是 -92^°（强左旋，故又称左旋糖 Levulose）！

1. 对映体（Enantiomers）、非对映体（Diastereomers）与差向异构体（Epimers）：

• 对映体：分子内所有手性碳原子的构型全部相反、互为实物与镜像关系的异构体（例如 D-葡萄糖与 L-葡萄糖；它们的 C2、C3、C4、C5 四个手性碳全部相反）。任一旋光异构体只有唯一一个对映体，其余 2ⁿ - 2 个异构体全都是它的非对映体。
• 差向异构体：差向异构体（Epimers）：在含多个手性碳原子的旋光异构体中，仅有一个手性碳原子的构型不同，其余手性碳构型完全相同的两个非对映体。在代谢中，差向异构体之间只需一步差向异构酶（Epimerase）即可相互转化。
• 必背三大己醛糖与两大戊酮糖的差向关系：在 16 种己醛糖中，生物界最常用的只有 3 种——D-葡萄糖（Glc）（C2、C3、C4、C5 羟基方向依次为“右、左、右、右”）、D-甘露糖（Man）（“左、左、右、右”）和 D-半乳糖（Gal）（“右、左、左、右”）。因此，D-葡萄糖与 D-甘露糖是 C2-差向异构体；D-葡萄糖与 D-半乳糖是 C4-差向异构体；而在戊糖磷酸途径中，D-核酮糖（D-ribulose）与 D-木酮糖（D-xylulose）是 C3-差向异构体。
• 命题大坑：D-甘露糖与 D-半乳糖是差向异构体吗？绝对不是！因为它们在 C2 和 C4 两个手性碳上的构型都不同，属于普通非对映体，在细胞内必须经过两步差向异构酶反应（以葡萄糖衍生物为中介）才能互变！

3D3D 糖类：单糖立体异构、成环变旋与化学性质交互查看 D/L 构型与差向异构体、Haworth 成环（上D顺β）、变旋现象与 β-D-吡喃葡糖全平伏键椅式构象、还原糖与糖磷酸酯↗

三、分子内亲核加成：环状半缩醛、异头碳与 Haworth 透视式

在黑板上画完直链 Fischer 式后，Vander Heiden 教授拿起手中的球棒分子模型问道：“如果我把一勺葡萄糖溶进水里，它真的像我刚才画的那样是一根直链醛糖吗？”

答案是否定的。在有机化学中，醇羟基（-OH）的氧原子带有孤对电子，极易对醛基或酮基的亲电羰基碳（C^δ+=O^δ-）发起亲核加成反应，生成半缩醛（Hemiacetal）或半缩酮（Hemiketal）。对于五碳及五碳以上的单糖而言，由于碳链骨架的键角弯曲，分子内部 C4 或 C5 上的羟基在三维空间上恰好直接撞在 C1 醛基（或 C2 酮基）的鼻尖上！于是，分子内羟基与羰基发生可逆的亲核加成，形成含氧原子的五元环或六元环：

• 吡喃糖（Pyranose）：成环后形成含有 5 个碳原子和 1 个氧原子的六元环，因结构类似于杂环化合物吡喃（Pyran）而得名。例如，D-葡萄糖由 C5-OH 进攻 C1 醛基，形成六元环的 D-吡喃葡萄糖；只有 5 个碳及以上的醛糖才能形成吡喃糖环，且醛糖在水溶液中吡喃环比呋喃环稳定得多（40^°C 时 D-吡喃葡糖占 99.9% 以上）。
• 呋喃糖（Furanose）：成环后形成含有 4 个碳原子和 1 个氧原子的五元环，类似于呋喃（Furan）。例如，D-核糖由 C4-OH 进攻 C1 醛基形成D-呋喃核糖；D-果糖当由 C5-OH 进攻 C2 酮基时形成五元环的D-呋喃果糖，当由 C6-OH 进攻 C2 酮基时则形成六元环的D-吡喃果糖。

当开链单糖的羰基碳（醛糖的 C1，酮糖的 C2）与分子内羟基加成为半缩醛（或半缩酮）环时，原本平面的 sp² 杂化羰基碳（非手性）突然变成了连有四个不同基团的 sp³ 杂化新手性中心！

异头碳与异头物（Anomeric carbon & Anomers）：单糖分子内成环形成半缩醛（或半缩酮）后，原来的羰基碳（醛糖 C1、酮糖 C2）转变为新的手性中心，称为异头碳（与两个氧原子直接相连）；仅在异头碳上构型不同的两个非对映异构体互称为异头物（即 α 型与 β 型）。

为了直观展示环状单糖，英国化学家 Haworth 提出了 Haworth 透视式（Haworth projection）：将糖环平面画成垂直于纸面，环朝向读者的一侧加粗，环氧原子放在右上方（六元吡喃环）或正上方（五元呋喃环），C1 放在最右侧，按顺时针方向依次编号 C1→C5。如何把 Fischer 式准确转换为 Haworth 式，并在考场上 1 秒钟秒杀 α/β 与 D/L 判读？请背熟下面这句黄金口诀：

3D3D 糖类：单糖立体异构、成环变旋与化学性质交互查看 D/L 构型与差向异构体、Haworth 成环（上D顺β）、变旋现象与 β-D-吡喃葡糖全平伏键椅式构象、还原糖与糖磷酸酯↗

四、变旋现象、椅式构象与“为什么自然界偏偏选中 D-葡萄糖作血糖？”

如果我们把纯的晶体 α-D-吡喃葡萄糖溶于水，立即放进旋光仪测定，会测得其初始比旋光度为 +112^°；而如果把纯的晶体 β-D-吡喃葡萄糖溶于水，其初始比旋光度只有 +18.7^°。然而奇妙的是，把这两杯溶液静置一段时间（或加微量酸碱催化）后，前者的旋光度不断下降，后者的旋光度不断上升，最终两杯溶液的比旋光度都停留在同一个恒定值——+52.6^°！

这就是著名的 变旋现象（Mutarotation）：新配制的单糖（或还原性寡糖）水溶液，其比旋光度随时间逐渐改变，最终达到一个恒定平衡值的现象。

为什么会发生变旋现象？因为半缩醛环在水溶液中并非死板一块，而是时刻发生着可逆的开环与闭环！变旋现象的化学本质是：环状的 α-异头物和 β-异头物在水溶液中通过极少量的开链醛式（Open-chain form）作为中间桥梁相互转化，最终达到热力学动态平衡。变旋现象是证明单糖在水溶液中以环状结构存在且能与开链式互变的铁证！

3D3D 糖类：单糖立体异构、成环变旋与化学性质交互查看 D/L 构型与差向异构体、Haworth 成环（上D顺β）、变旋现象与 β-D-吡喃葡糖全平伏键椅式构象、还原糖与糖磷酸酯↗

用简单的一元一次方程就能算出平衡体系的组成：设 α 型占比例 x，β 型占 1-x，则 112x + 18.7(1-x) = 52.6，解得 x ≈ 36%（约 1/3），1-x ≈ 64%（约 2/3）。精确测表明，在 D-葡萄糖的水溶液平衡体系中，约 1/3（～ 36%）为 α-D-吡喃葡萄糖，约 2/3（～ 64%）为 β-D-吡喃葡萄糖，而开链醛式及呋喃糖形式合计不足 0.024%（因此游离葡萄糖的普通醛基性质表现得极不明显）。

此时，Vander Heiden 教授在课堂上抛出了全节最精彩的两个追问：

追问一：为什么在葡萄糖水溶液中，β 型占了 2/3，比 α 型（1/3）稳定得多？

追问二：在 16 种己醛糖中，为什么进化偏偏选中了 D-葡萄糖作为所有生命通用的血糖与核心储能单体？

要解开这两个谜团，必须跳出平面的 Haworth 式，看清吡喃六元环在三维空间中的真实面貌——构象（Conformation）。

正如环己烷一样，六元吡喃糖环绝不是平面的，它可以采取椅式构象（Chair conformation）或船式构象（Boat conformation）。由于船式构象存在严重的立体拥挤和重叠张力，吡喃糖在水溶液中主要采取能量最低的椅式构象（注意：王镜岩教材提到的“椅式与船式之比约 1000:1”是环己烷的数据，而非葡萄糖的实测值，切勿张冠李戴）。在椅式构象中，环上的 12 个化学键分为两类：6 个平行于环对称轴、直立上下的直立键（Axial bond，简称 a 键），以及 6 个朝向环四周外侧伸展的平伏键（Equatorial bond，简称 e 键）。任何体积比氢原子（-H）大的取代基（如 -OH 和 -CH_2OH）如果处在直立键（a 键）上，都会与同侧间隔碳上的直立键基团发生强烈的 1,3-二直立键空间位阻排斥（Steric hindrance）；只有处在平伏键（e 键）上时，才能最大程度向四周散开，互不干扰！

现在，让我们把 β-D-吡喃葡萄糖 放进椅式构象Ⅰ（⁴C₁）中：

你会震惊地发现，它的 C1-OH、C2-OH、C3-OH、C4-OH 以及庞大的 C5-CH_2OH，全部五个非氢大基团完完全全处在空间位阻最小的平伏键（e 键）上，没有任何一个大基团处在直立键上！

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~00:00MITOCW | 12. Carbohydrates/Introduction to Membranes

~00:02PROFESSOR:

~00:02OK. Great. Now let's dive into the material today. So today's topic is carbohydrates as well as an introduction to membrane structure. And from the very first class, I believe Professor Yaffe talked to you about the four main classes of biological molecules. So proteins and amino acids, you've spent a lot of time talking about that. Nucleic acids is coming up. And the other two are carbohydrates or sugars and lipids. Today, we're really going to focus most of the lecture on carbohydrates, what they are, structure, something about nomenclature. And then at the end, we're going to talk a little bit about lipids and basic membrane structure. Now carbohydrates and lipids are really critical energy storage molecules for cells. And when we talk about metabolism-- the most interesting part of this course-- after spring break, we're going to delve into this in a lot more detail. But sugars are also very important to understand nucleic acid structure. Turns out, membranes are essential for signal transduction. Those are the two major topics that Professor Yaffe is going to talk about for the rest of his time. And so this year, we're going to try something different and have me introduce at least some of these topics now, which will deal with some redundancies that otherwise might have existed and maybe set it up better for him to discuss some of the lectures coming up. Now today's lecture is unfortunately topically a little bit disjointed. But it still has important information about biochemistry. And it will help us all speak the same language, both for the upcoming lectures from Professor Yaffe as well as things that I will start off with when I come back. And it's a nice way to ease back in after the exam. OK. So what is a carbohydrate or a sugar? So let's break this down, and so it's carbohydrate. So a carbohydrate is effectively carbon in some ratio with water. So all carbohydrates have the same chemical formula, Cn and H2On. And so there are some deviations from this in biology. Sometimes, you can introduce a heteroatom, phosphate, sulfur, nitrogen, et cetera. Technically, these are not carbohydrates, although they're often lumped together with carbohydrates. And why would nature do this? Because it changes some of the chemical properties that can be useful for either structural or signaling reasons. You may encounter these later-- certainly, in other classes. But we're not going to talk much more about that today. We really just kind of focus on the base carbohydrate, Cn H2On structure. Now carbohydrates come in different forms. And so they can come as single units with that structure. These are so-called monosaccharides. Or these single units can come together to form various polymers. And those polymers could be two units, so-called disaccarides-- so two sugars stuck together. Or many units, and so sometimes, those are referred to as oligosaccharides or polysaccharides. And there's really no clear distinction between a few chains together, oligosaccharides, many polysaccharides. They're somewhat used interchangeably.

~03:28Now you guys have almost certainly heard of many of these things. So what's a monosaccharide? So a good example of that is glucose. So glucose is, of course, the main sugar that exists in your blood. What's a disaccharide? So a common one is sucrose. So sucrose is a disaccharide-- so two sugars stuck together, glucose plus fructose. You've probably heard of both of those things before. Sucrose is, of course, table sugar. It's what you would have mixed into your coffee if you had that this morning. And an example of a polysaccharide is starch-- so what's in a potato. All right. Now clearly, these are all sugars. They're all relevant to a human diet. You would never confuse-- you've probably not knowingly tasted glucose. It's not particularly sweet. Sucrose is much sweeter. And a potato is not necessarily sweet at all. Yet, this really points out that how these sugars are built, the different structures matter a lot. They matter for things like how you taste them in your diet. And how carbohydrates are built also matters a lot for all kinds of aspects of biology, which is why it's somewhat important to at least understand some of what we're talking about-- a common language about how to describe these molecules. OK. So the simplest biological sugars have three carbons that are least commonly used. These are referred to as trioses. And if we take the general formula, C3 H2O3, and we say, what are two ways that we can satisfy that formula? There's two main ways we can do it. One is like this. So if you add up-- count all the carbons, hydrogens, and oxygens, you will see that that is C3 H2O3. This molecule is called glyceraldehyde. OK. And the other way we can do this is like this. OK. Again, three carbons, three waters. If you add up all the atoms, this molecule is called dihydroxyacetone. And these are sugar-- same chemical formula, different chemical structure. That has a term. That's called an isomer. And it turns out that we can chemically interconvert these molecules in the following way. And so if we carry out this chemistry, we will get this intermediate.

~05:58OK. And that will allow you to interconvert glyceraldehyde-- this aldehyde-- with this ketone, dihydroxyacetone. And the enzyme class that carries this out is a class of enzymes called isomerases. And this is exactly the chemistry that that enzyme would use to interconvert these two forms of this triose, this three-carbon sugar molecule. All right. Now if we look at dihydroxyacetone, there's no stereocenter here. What do I mean by a stereocenter? That's a carbon that has-- reminder from 512-- carbon that has four different non-equivalent substituents around it. However, if you look at glyceraldehyde, that carbon in the middle is a stereocenter-- four non-equivalent groups around it. And so there's two ways that I can draw glyceraldehyde. I can draw it like this. Or I could draw it like this. All right-- so two different ways. So the one on the left here is D-glyceraldehyde. And the one on the right is L-glyceraldehyde. OK. And I know that this is review. Some people are good at seeing these things. Some people are not. I brought a couple of models here. Here's this-- the blue and the brown are two stereocenters with four different constituents on them. No way you can twist these around to make them identical molecules. Why does this matter? Well, enzyme-active sites are going to fit this molecule different than this molecule. And this is why these stereoisomers matter so much for biology. It's also something that's really hard to accomplish.

~07:38If you think about how do you actually generate stereoisomers if you were in an organic chemistry lab-- really hard. But biology does this all the time. And the real reason is because it's enzymes that ultimately catalyze these interconversions. And different stereoisomers will fit differently into enzyme-active sites. Now the way that I've been drawing these sugars is a convention called a Fischer projection. And when drawn in this way, the convention is that-- so you put the carbonyl towards the top. If the OH group, the alcohol, is pointing to the right, that's D. If it's pointing to the left, that's L-- and so D, pointing to the right, L, pointing to the left. Like amino acids, biology has chosen one stereochemistry for most biological sugars because, of course, enzymes act on them. And so in biology, it's D, sugars. This is in contrast to L, amino acids. And so if you can remember that sugars are D, you know that amino acids are the opposite. Or if you remember that amino acids are L, you can remember that sugars are the opposite. OK. All right. So dihydroxyacetone is really the only sugar with three or more carbons that doesn't have a chiral center. Everything else will. And so if I go to a four-carbon sugar-- draw a couple of them here. OK. So here's C4 H2O4 drawn with a ketone. If you look at this, this is a chiral center. OH group is pointing to the right. This is a D sugar. If I had drawn it with the OH group on this side, it would be an L sugar. All right. If I draw this sugar as a different isomer, this time with an aldehyde, now, we encounter a bit of an issue because now I have one, two stereocenters. OK. So if there's two stereocenters, that means there's 2 to the n ways that I can draw this as a stereoisomer. And so you can see that this could get really complicated very quickly. Now this sugar obviously is a D because I drew both OH groups pointing to the right. But you can imagine, I could draw one this way or one that way. And so how do if it's a D or an L sugar? And so the convention is that whether or not a sugar is designated as D or L refers to the stereocenter that is furthest away from the carbonyl. And so this is the relevant stereocenter that says it's a D sugar. So what do I mean? I can draw. And then any other sugar would have a different name. And so what do I mean by that? Let me draw this a few different ways. And so the sugar that I've drawn here is referred to as D-erythrose. All right. If I draw it, now the OH group on the carbon furthest from the carbonyl pointing to the left, so this would be L-erythrose. And if I draw it differently by altering the stereochemistry of this carbon, now, it has a different name. And so this carbon is D-- or this sugar is D-threose. All right. Makes sense. If I flip the OH group to this side, it would be L-threose. All right. So lots of possibilities. Turns out nature only uses a subset of the stereoisomers and makes them relevant for biology. For example, Derythrose is something that you will encounter when we talk about metabolism later in the course. D-threose, as far as I know, is not used in biology. I would never say, it's never used in biology. Never bet against biology. It can do absolutely everything. There's always an exception somewhere. But, in general, D-threose is not something that really exists, at least commonly in nature. All right. So if we go through, and you look at all of these different sugars that I've drawn, you can see that they either have an aldehyde, or they have a ketone somewhere in the molecule. All right. So if you have an aldehyde, these sugars are generically referred to as aldoses. All right. And if you have a ketone, an internal carbonyl, these are generically referred to as ketoses.

~12:24Now you might say, as you start getting to longer and longer sugars, you can put the ketone anywhere along the sugar, and there would be a ton of different possibilities. But it turns out ketoses always have the carbonyl as the second carbon in from the end. The reason for that is because biology, as you will see when we talk about metabolism, interconverts these via isomerase reactions. And so you can't use an isomerase reaction to interconvert a ketose and an aldose unless the ketone is one carbon away from the end of the sugar. And so this fact really limits some of the diversity of ketoses that can actually exist in nature. Now most important biological sugars, at least the most common ones, end up having six carbons or five carbons. And these are referred to as hexoses or aldose-- or pentoses. Sorry-- so much nomenclature. OK. So sixcarbon sugars are hexoses. Five-carbon sugars are pentoses. And if we just talk about the hexoses, you're very familiar with a couple of them. And so one that we mentioned earlier, blood sugar-- glucose looks like this. So this molecule is D-glucose. It's a D sugar because the stereocenter here furthest from the carbonyl-- OH group points to the right. So it's a D sugar. It's an aldose because it has an aldehyde. All right. And it's a hexose because it has 1, 2, 3, 4, 5, 6 carbons. And what makes it glucose is the stereochemistry of these other sites that end up being-- this is the molecule glucose. Now if I carry out that isomerase reaction that I showed you earlier, it'll give me this intermediate. Let me draw the whole thing. Then I interconvert that aldose. Now, it becomes a ketose. This is D-fructose-- another common sugar that certainly is in the news a lot. D sugar because the stereocenter for this from the carbonyl points to the right, makes it a D sugar. It's a ketose. It's a hexose. This organization of other stereocenters is what makes it D-fructose. All right. Now if we go through and we count these, there's four stereocenters in glucose, three stereocenters in fructose. That means 2 to the n. There are 16 ways I can make ketose aldose, eight ways I can make a ketose-- I'm sorry-an aldose hexose, eight ways I can make a ketose hexose. Among those, that's even 12 different ways that I can have D hexoses that are a ketose or an aldose. Could be very, very complicated. But it turns out, fructose is the only D hexose ketose that really is relevant for nature. There's only two other molecules related to glucose-- only two other hexose aldoses that are sugars used in nature. You've probably heard of these as well. I'll draw them to illustrate a different point. OK. So this molecule is galactose, an important sugar found in milk. All right. If I draw a glucose next to it again, glucose-- so these two sugars differ here, the stereochemistry there. So they are, I guess, isomers of each other. There's a special name for it I will get to in a second. So that's how galactose is related to glucose. The other major aldose hexose is this one. This is D-mannose. This differs from glucose here-- that carbon at that carbon. By the way, by convention, the way that you number carbon in sugars is you start with the end that's closest to the carbonyl, either the aldehyde or the carbon one up from the ketone, so this would be carbon 1, 2, 3, 4, 5, 6. So galactose differs from glucose at carbon 4. Glucose differs from mannose at carbon 2.

~16:36These isomers have names in relation to each other. And so two sugars that differ by one part of stereochemistry-- so galactose to glucose or glucose to mannose-- are called epimers. And these can be or converted by enzymes called epimerases. We'll talk about how these work later in the course. And so glucose is an epimer of galactose. Glucose is an epimer of mannose. Mannose is not an epimer of galactose because mannose and galactose differ at both carbon 2 and carbon 4-- their stereochemistry. Why is that relevant? Because if you're going to interconvert galactose and mannose, you would have to do it in two steps, two different epimerase reactions to interconvert those two sugars. All right. Great. So we've discussed now all of the major hexoses that nature uses. We've discussed all the major trioses that nature uses. The other major length of sugars that ends up being important in biochemistry is the five-carbon sugars, the pentoses. And so I want to mention a couple pentoses and do so in a way that will allow me to basically solidify some of the nomenclature that I've gone through. And, of course, one of the five carbon sugars-- ribose-- is really critical to nucleic acid structure, which is one of the reasons why we're talking about this at this point in the course. OK. So this is a pentose-- 1, 2, 3, 4, 5-carbon sugar, D-ribose. All right. It's a D sugar because the stereocenter furthest from the carbonyl points to the right. It's an aldose because it has an aldehyde group. I can act on this with an isomerase. I'm not going to draw out the isomerase reaction again. It's exactly what I drew before. If I did this, now, I turn this into a ketose. Pentose because it's five carbons. Ketose because as a ketone. D sugar because the stereocenter furthest from the carbonyl points to the right. This has a name of D-ribulose. All right. And it turns out that there is an important epimer of D-ribulose that's found in nature. The epimer changes the stereochemistry at carbon 3-- carbon 1, 2, 3, 4, 5. That's carbon 3. And so if this was acted on by an epimerase that did that, you get this sugar, also a pentose, also a ketose, a D sugar, but an epimer of ribulose. It's called D-xyulose. It's an epimer because xyulose ribulose are epimers because they differ by stereochemistry only at one position. I'll just say, right off the bat, you should not memorize names of sugars and their structures. These are things you can look up in books. The point of going through all this is just to expose you to some of the nomenclature, remind you about stereochemistry. I realize, these are basic things. Many of you have already encountered this. Some of you find this very easy. Some people find these sort of spatial things more difficult. This is very well-reviewed, though, in textbooks or other places online if you need to look it up. But the key thing is just to remember this nomenclature because it'll make it easier for us to talk about sugars later in the course. All right let's take a short break, so I can get some board space back. And then we'll build off some of these concepts in a minute. I've been drawing all of these sugars as straight chains. But you probably know, from high school or from looking at DNA or RNA that the ribose there is not a straight chain but, instead, forms a ring. And, in fact in solution, particular aqueous solution, sugars, particularly five carbons and longer almost always exist as rings. And there is a very clear reason for this. And you probably remember, from 512 Organic Chemistry that alcohols will react with aldehydes and ketones in solution.

~20:58And so here, I have a model of glucose and fructose if you want to come and play with them. And so if you look at this, and you just look at the model, you see that this oxygen, right here, this alcohol, in space, is very close or can be moved to be very close to this aldehyde here on the end of the molecule. Or the same thing here with fructose. Here's a alcohol very close in space with this ketone. All right. So what happens in this situation? Well, if you have-- this is a review of organic chemistry. OK. So here's any generic aldehyde. Here's some alcohol. OK. So those things react. You end up with this so-called hemiacetol. Or the same thing if I do it with a ketone and an alcohol-- now you get this hemiketal. OK. Now given that you have an alcohol reacting with a carbonyl, an aldehyde, or ketone on the same molecule, well, what happens is you effectively get a ring with oxygen being one of the components of the ring. And so if we draw this for glucose-- so this is D-glucose. All right. And so if the alcohol here on carbon 1, 2, 3, 4, 5 interacts with the aldehyde on carbon 1-- you can play with the model and see that that is the one that's close in space-- you now end up getting this. This, where you get a ring between carbons 1 and 5. We can number the rest of them-- 2, 3, 4, 5, 6. All right. Or if I turn this molecule so that you can now draw it in a slightly more chemically proper way, now, you basically have-- this is carbon 1, 2, 3, 4, 5, 6. So carbon 5, oxygen from carbon 5 now bound to carbon 1 gives you this six-membered ring structure. The six-membered ring structure is reminiscent of an organic molecule called a-- looks like that-- called pyran. And so this six-membered ring in a sugar is also referred to as a pyranose. All right. So that's the first thing. The second thing is that this whole business can be very tedious to draw. In fact, it's very tedious drawing sugars in general, as I'm sure you would agree with me if you're taking notes during this lecture. And so oftentimes, these pyranoses, like glucose, are drawn with shorthand. And the shorthand is as follows, where I basically represent the OH groups as simply lines. And so this is another shorthand to draw that pyranose form of glucose-again, carbon 1, 2, 3, 4, 5, 6. Now the last thing is you can see I didn't draw the OH group there on carbon 1. And that's because by making this pyranose ring, if you look at carbon 1, I have now generated a new stereocenter. And so carbon 1 now has four non-equivalent groups on it, which means I could draw the OH group, that carbon 1, in two different ways. And so this is carbon 1. I could draw it such that the OH group points down. Or I could draw it such that the OH group points up. And those are two different molecules. And so there's a naming convention for this too. And so if the OH group points down using this way of drawing the molecule, it's called alpha. If the OH group points up, it's called beta. All right. And so this alpha versus beta ends up being structurally different because it puts, basically, that OH group pointing in a very different position in space. So if I make a ring here with glucose, and the OH group is pointing here versus there-- very different position in space. And this has implications for how you build bonds for disaccharides and polysaccharides that make structural differences. And we'll cover this a lot when we get to metabolism.

~25:24But it should be very clear now that glucose, if you just take a solution of glucose and put it in water, it is not one thing. There's actually multiple different forms it can have. It could be as I drew it up here with the OH group pointing down. This would be alpha D-gluco-- because it's glucose-- pyranose, because it's in the pyranose ring form. I could have drawn it with the OH group pointing up-- different molecule. That would be Beta Dglucopyranose. Or it could just be the open-chain D-glucose that I was drawing earlier. All right. All of those are perfectly legitimate ways for glucose to exist in solution. All right. Now it turns out, that in reality, about a third of it in solution is this. Two thirds is that. And a trace amount is this. OK. And that has to do with just what is more favorable forms or not. But which form it's in actually matters for structural reasons, as we'll see later in the course. Now the final complexity is that this ring is not flat. So if I actually take glucose here and make a form of it-- so here's my glucose molecule. There's no way for me to make this completely flat, say, like benzene. And so there's really two different pyranose confirmations that can be formed. I'll try to draw them, but they're harder to draw. Here's a overhead that you can look at if it's easier. But basically, you can have it form this so-called boat form or this so-called chair form. So there's the boat versus the chair conformation. Glucose, turns out, prefers the chair conformation. And there's an interesting thing that comes from this because if you take beta D-glucopyranose, it turns out of all the hexoses that exist in possible hexoses that exist, the form of a hexose aldose that best spreads out all the hydroxyl groups is beta D-glucopyranose, the more common one in solution. Why does this matter? Well, because if this has this reactive aldehyde bound up in this stable ring structure, it's less likely to react with other aldehydes in the cell. And so this is likely why nature chose D-glucose as the most common storage sugar-- why it's sugar in your blood-- because it's the most stable hexose that's out there. And it's not just a random reason that nature picked this one, but actually because of real chemical stability issues for why it's there. All right. Now I want to mention that ketoses also can form rings. And I'm going to use this as an example to show you that a ketose-- so here's fructose. So this is D-fructose. And so it turns out, this can form two possible rings. It can form a five-membered ring or six-membered ring. So how do I form a five-membered ring? So if I take the carbon here from carbon 1, 2, 3, 4, 5, the hydroxyl from carbon 5, form a ring there. Now, I get this molecule. So there's 1, 2, 3, 4, 5, 6. OK. Or if I now turn this so that I draw it in the way you're probably more used to seeing it, I'm going to use the shorthand here. So this here would be carbon 1, 2, 3, 4, 5, 6-- hydroxyl from carbon 5, forming a bond to carbon 2. That creates a new stereocenter at carbon 2. OH group is pointing up. So this is a beta sugar-- OH group pointing up. So this here, as I drew it, would be beta D-fructo and this is a furanose. Why is that? Because the organic molecule that's a five-membered ring with an oxygen in it is a furan. And so the five-membered ring is referred to as a furanose-- beta D-fructofuranose. If I'd drawn it with the OH group pointing down, it would be alpha Dfructofuranose. OK. There's another possible ring I can do. And instead, I take the hydroxyl from carbon 6 and do that-- form a ring. Now I'm going to form a six-membered ring. We have carbon 1, 2, 3, 4, 5, 6.

~30:05So if I now turn this-- so this, here, now I drew the OH group pointing down. So it's an alpha. If I drew it up, it would be beta. So this is alpha D-fructofuranose-- six-membered ring version of fructose, five-membered ring version of fructose. So lots of non-equivalent ways I can draw fructose. And it turns out, these actually matter. And they matter for real things that probably matter to you. And so I brought with me here two different sweeteners. This is corn syrup. This is honey. Has anyone ever-- I'm sure most of you have had honey. Has anyone ever tasted corn syrup? Come on. Someone's tasted corn syrup. Is a sweet? Which one's sweeter? Honey, by far-- much, much, much sweeter. I used to have it so you guys could come up and taste them. But I couldn't come up with a way to do that in a sanitary way. So I gave up on it. But nonetheless-- much sweeter than that. It turns out, these are not pure fructose. They're a combination of sugars. But their composition is actually, from a chemical standpoint, similar amounts of fructose in each one. And it turns out that honey is beta D-fructopyranose. Whereas corn syrup is beta D-fructofuranose. All right. So same sugar, different structure-- one's a furanose, one's a pyranose-- tastes very differently to you, one being much more sweet, one being much less sweet. OK. Now, of course, I also want to talk about ribose because it's the thing that you guys are going to talk about the most next because it's in nucleic acids. And so just as a reminder, here's ribose. It's a aldose and a pentose. So this is D-ribose. Ribose, as from high school, forms five-membered rings. That's because it links alcohol on carbon 4 to the aldehyde on carbon 1. And that gives you this ring structure. Now number of carbons-- 1, 2, 3, 4-- alcohol on carbon 4, forming a ring to the aldehyde on carbon 1. This is carbon 5. And the way I drew it, OH group pointing up-- so this is beta D-ribofuranose would be the proper way to have it. And so when you guys talk about this in DNA, well, the base is going to be linked to carbon 1. It's going to replace that hydroxyl group-- the beta form of the hydroxyl group with the nitrogen. And you're going to talk about bonds being from the 5 prime or the 3 prime end. Those are those hydroxyl groups. That's where 5 prime and 3 prime comes from because those are the 5 and the 3 position on ribose. Great. Perfect. All right. So we will return to carbohydrates in great detail. We'll discuss how we combine different carbohydrates, different monosaccharides to make disaccharides and polysaccharides, how these different structural properties end up mattering for things like energy storage as well as to produce various structural molecules that can be important for different cells in organisms. But for the remaining part of the class today, I really want to shift topics to now discuss a completely different class of biomolecules. And that's lipids. So as I said earlier, the reason we're going to do this is because coming up for Professor Yaffe's lectures, lipids end up being really important molecules for various aspects of cell signaling. They're also very important for energy transduction, which is why I'm talking about them as well. We will spend a lot of time later talking about lipids in great detail. But really, what I want to focus on today is how lipids are used to create membranes. That is, barriers that really separate the outside world from the inside of cells, things that make compartments within cells, and these things also end up being surfaces where you can have key things happen. So you reduce spatial complexity if you go to a 2D surface versus a 3D surface, which is part of why they're so important in signal transduction.

~34:38But to talk about these, I have to introduce, first of all, what is a lipid? So lipids are a class of molecules. You think of them as fats. That's how they fit in the nutritional standpoint of what we'll all talk about. But first, I want to give a general definition of what a lipid is. Now there's lots of different classes of lipids. And we'll cover these later in the course. But in general, at the highest level, almost all lipids consist of two pieces-- consist of something called a fatty acid that's esterified to an alcohol. So what is a fatty acid? Well, a fatty acid is really any molecule that has a carboxylic acid group. So there's a carboxylic acid. And then hooked up to that carboxylic acid is a whole bunch of saturated hydrocarbons- alkyl chains. Oftentimes, fatty acid would be drawn like this because of all the different saturated chains. So acid group on one end, really greasy alkyl chain on the other. And it turns out that this can be esterified to an alcohol. So here's just a generic alcohol. And as you certainly remember from organic chemistry, I can use an alcohol and an acid to form an ester. And so then that would be-- so now, I have, basically, this ester bond. And this is esterification between some lipid species and a fatty acid, in general, is the general thing that leads to a class of molecules known as lipids.

~36:19Now why this is particularly useful for biology is that this long, greasy alkyl chain is not water soluble. So if you take oil from your cabinet-- canola oil, or olive oil, or whatever, and you pour it in water, what happens? It doesn't mix together. You get these globs of oil floating in the water. And so if you want to form a barrier between two aqueous compartments, a way to do this is to have, basically, a hydrophobic layer-- a membrane-- basically separate the two aqueous compartments, the inside and the outside of the cell. Now if I take a lipid, and the lipid, I form this ester, and it doesn't have a charged group anywhere on the molecule, this is sometimes referred to as a neutral lipid. So what do I mean by neutral lipid? It's basically taking this, which is intrinsically a fatty acid, is intrinsically a polar molecule. It has this carboxylic acid on the end of it. But if I form an ester linkage to an alcohol, and the alcohol has no charge on it, now, it's just this really greasy molecule. And it turns out, that's exactly what is in your olive oil or in your canola oil in your cabinet. It's basically a bunch of fatty acids-- long alkyl chains esterified to non-charged alcohols, which makes this neutral lipid, this greasy molecule, which is great for energy storage in plants, something we'll talk about. But if you want to mix it together with water, it doesn't do very well. So if you go to make salad dressing, and you pour your oil in with your vinegar, the aqueous thing, you get a bunch of droplets. You don't get a solution. And if you think about it, that's not very good at generating an interface between two compartments. You just get a bunch of droplets. You actually don't get an interface between two compartments. So if you want to make an interface, you have to do something different. You need to have a charge group, like on this fatty acid, that's going to be happy sticking towards the aqueous side and then a hydrophobic portion that's going to be happy sticking to form a barrier. Effectively, that's what soap is. So if you want to wash your hands and use canola oil, it doesn't work very well, right? But what happens if you have a bunch of greasy stuff in the water, and you take a drop of dawn dish detergent and drop it in the water?

~39:12You get this immediate barrier that forms across the top as you get this film where you basically align up all these charged hydrophilic parts to the aqueous pieces and all of the greasy parts to the hydrophobic side. So you end up having this nice charged molecule with this long hydrophobic part. So you have a hydrophilic end and a hydrophobic end. And that ends up being very useful as a way to form an aqueous hydrophobic interface. And this is exactly how soap works, as I'm sure you've learned in other classes. As an aside, you know how you make soap? You basically boil the neutral lipids in lye. So boiling it in base, if you remember from organic chemistry, will break the ester linkage. And now, you have these molecules. And that's how you make soap. All right. Now the way biology does this is they actually assemble lipids that effectively have that same property, where, basically, they have a head group from the alcohol that's charged with a hydrophobic group contributed from these fatty acids. And this is what allows things to assemble into membranes. And so membranes are largely made up of-- and certainly, for the purposes of the upcoming lectures-- so-called phospholipids. So what is a phospholipid? Well, on a phospholipid, the alcohol part of the lipid is derived from a molecule called glycerol. So glycerol looks like this. OK. So this is glycerol. All right. 3-carbon molecule-- three alcohols-- actually, very similar. If you look back in your notes to dihydroxyacetone, the difference between glycerol and dihydroxyacetone is carbon 2 was an alcohol here before it was a ketone. Turns out, that's where glycerol comes from-- dihydroxyacetone getting turned into glycerol. And basically, what a phospholipid is that you esterify a fatty acid to two of the alcohols and another phosphate and charged alcohol to the other hydroxyl group. So what do I mean by that?

~41:24So like this-- so here's ester linkage number one. I'll draw it this way just for ease. This is the carbon 2. Here's ester linkage number two. So that's a fatty acid esterified to 2 and 3. And then you now make an ester to a phosphate. So there's a phosphate at carbon 1. And then you make another ester linkage to another alcohol where R equals alcohol. And that alcohol also turns out to be charged. And so the most common phospholipid membrane lipid is a molecule called phosphatidylcholine, which is that structure with the R group being this alcohol. And so this is choline. OK and so phosphatidylcholine is basically that structure. OK. So this is phosphatidylcholine drawn out in all of its glory. So you have this hydrophilic end. Here, you have a hydrophobic end. That's good. This can point towards the aqueous side. This can point towards the greasy side and make a nice interface. And so another common word for phosphatidylcholine-- an old word for it-- is a molecule called lecithin. Anyone read the labels on the side of your food ever? In fact, there's an old commercial that makes fun of this. Soy lecithin? What's that? I don't want that in my ice cream. Well, what is lecithin? It's phosphatidylcholine. It's basically, why is this done? Well, it's commonly added to ice cream because what is ice cream? And it's an emulsion between fat, the cream, and sugar, which is pretty aqueous solubles, you might guess, from looking at what we drew earlier today. And so by putting phosphatidylcholine in your ice cream, you basically stabilize this emulsion between the aqueous and the fat face. So this is why it's added to lots of food. And in fact, it says, soy lecithin, an emulsifier. That's why it's added.

~43:27You can do this yourself. This is a common trick that many cooks know. So if you make salad dressing, you put a little mayonnaise in your salad dressing. Why do you do that? Well, mayonnaise has eggs in it. Eggs have a lot of phosphatidylcholine. That mayonnaise that you add into your salad dressing basically stabilizes that emulsion between the oil in the vinegar and helps it be more stable and distribute better across your lettuce. I think my favorite example of this is, who's made chocolate chip cookies? Everyone's done this, right? OK. So how do you make chocolate chip cookies? So you take butter and sugar, all right, fat, and something that's very non-- very polar, aqueous soluble sugar. And you try to mix them together. You have to cream it, right? And that takes forever. It's a pain in the butt. It doesn't come together very well. And so you get lazy, and you put the egg in there. And then it all comes together really, really easily. So why is that? The egg is the emulsifier that actually brings it together. Whereas trying to get the butter and sugar to make your cookies nice and fluffy is a lot harder because you're really trying to get these two things to come together. And it's basically this exact chemistry that's taking place when you're doing that act of cooking. All right. Now phosphatidylcholine is not the only phospholipid found in membranes. But I'll just quickly mention what a couple of the other ones are. So it turns out you have two other major phospholipids by abundance and then another phospholipid that's really important for signaling. So I'll list them here first. So there's phosphatidylserine, phosphatidylethanolamine These, it turns out, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine is what makes up the majority of the phospholipids in cell membranes. And then there is a much more minor phospholipid that's important for signaling, which is phosphatidylinositol. So what are these? So serine, ethanolamine, and inositol are just, like choline, different alcohols that can be esterified to the phosphate in exactly the same way I did for phosphatidylcholine. And so what's phosphatidylserine? So if you remember, this is-- so this here is the amino acid serine. I'm sure you remember that. If we esterify that alcohol to the phosphate, there's phosphatidylserine. What's ethanolamine look like? So ethanolamine is this alcohol. So that's ethanolamine. Esterify the alcohol to phosphate. Esterify that phosphate to glycerol-- phosphatidylethanolamine. And the last one is inositol. So what is inositol? Well, inositol is a six-membered hydrocarbon rain where all of the carbons have an alcohol on them. And so if I esterify one of these alcohols on inositol, versus a-- if I esterify one of those alcohols to a phosphate to make phosphatidylinositol, turns out that that ends up being a really useful lipid to make for signaling. And you'll hear a lot about this in a couple of contexts, I believe, from Professor Yaffe later in the course. All right. So this having these general membrane structure or this general phospholipid structure, where you have this hydrophobic part of the molecule and the hydrophilic part of the molecule is really what allows these lipids to come together to form these membrane bilayers that are often drawn like this. And so when I'm drawing it like this, basically, what those two wavy lines represent-- those are the hydrophobic fatty acid parts, and this is the hydrophilic so-called head group.

~47:24That's the charged alcohol stuck on to the end of the-- to the phosphate on the glycerol. And these, of course, assemble such that all of the hydrophilic head groups face an aqueous compartment on either side. OK. And then you have this nice hydrophobic portion that is a membrane. And that is what can create a barrier between two different compartments in cells. Now just to put this into context for the protein structure stuff that you learned about-- so when you learn about all the different ways you have protein structure, of course, you have amino acids that are hydrophilic, amino acids that are hydrophobic. If you have a protein that's floating in solution, it has all the hydrophilic parts on the outside and the hydrophobic parts in the middle. But you can imagine that you can also have proteins that assemble to sit such that they span the membrane-- so hydrophobic parts that interact with the hydrophobic part, the inner part of the membrane-- hydrophilic parts on either side. These can form channels. These can form all kinds of ways to move stuff across membranes. Or you might think that you might have a protein that does something like that-- hydrophobic part on one end, hydrophilic part on the other, and really floats on the surface of a membrane. And you will see that the way cell signaling works is that you basically have lots of protein complexes that will assemble that membranes-- two dimensional space. Those membranes, as well as some of the membrane lipids, can then act as messengers that allow cells to carry out various signals. And I will leave that to Professor Yaffe to talk about. All right. So I will also come back and discuss membranes in a very different context in the context of metabolism during the second half of the course because it turns out, a lot of energy transduction and the way cells really store energy also ends up being really important with respect to membranes. All right. You guys are lucky. You get out a little bit early today. This is the first time that we've done the lectures this way, so I don't have my timing quite right. But I will see you guys again for metabolism after spring break.

1.2单糖的化学性质、糖苷键与重要二糖：为什么蔗糖和海藻糖是非还原糖？

一、单糖的化学反应与重要衍生物：围绕羰基与羟基的六大反应

单糖分子兼具羰基（醛基或酮基）与多个醇羟基，其全部化学性质都围绕这两类官能团展开。在考研生化中，有六项反应与三类衍生物是高频命题点：

1. 酸碱稳定性与稀碱中的异构化（烯二醇中间物）：
   单糖对稀酸相当稳定（加热强酸则会脱水生成糠醛或羟甲基糠醛，可与酚类如 α-萘酚发生 Molisch 紫环反应鉴定糖类）；但在稀碱溶液中，单糖极易通过烯二醇中间物（Enediol intermediate）发生酮-烯醇互变异构。例如，将 D-葡萄糖置于稀 Ba(OH)₂ 溶液中放置数天，C1 和 C2 之间会形成双键并丢失 C2 的手性，重新异构后变成 D-葡萄糖、D-果糖和 D-甘露糖 三者的平衡混合物（Lobry de Bruyn–van Ekenstein 转化）。
2. 温和碱性铜试剂氧化与还原糖（Reducing sugars）：
   在 MIT 7.05 L19 课堂上，Vander Heiden 教授详细拆解了生化实验室最经典的糖定性检测——Fehling 试验（斐林试剂）与 Benedict 试验（本尼迪克特试剂）。Fehling 试剂含有酒石酸钾钠、NaOH 和蓝色的 CuSO₄（Cu²⁺）；Benedict 试剂则用柠檬酸和 Na_2CO3 代替酒石酸钾钠和强碱，试剂更稳定，曾广泛用于临床尿糖与血糖半定量检测。

• 反应机制：当把糖与碱性 Cu²⁺ 溶液共热时，只要该糖分子拥有游离的半缩醛（或半缩酮）羟基，它的环就能打开（Access the open-chain form）暴露出游离醛基（或酮基）。游离醛基随即被氧化为羧基（醛糖酸），同时将蓝色的 Cu²⁺ 还原为砖红色的氧化亚铜（Cu_2O↓，即 Cu⁺）沉淀！
• 还原糖（Reducing sugar）：分子中含有游离的半缩醛（或半缩酮）羟基，在水溶液中能开环暴露出游离醛基或酮基，从而还原温和氧化剂（如 Fehling 试剂、Benedict 试剂中的 Cu²⁺ 或 Tollens 试剂中的 Ag⁺）的糖类。
• 为什么果糖没有醛基，却也是还原糖？ 因为 Fehling 和 Benedict 试剂都是碱性溶液！果糖（酮糖）在碱性受热条件下，会迅速通过烯二醇中间物异构化为葡萄糖和甘露糖（醛糖），从而照样把 Cu²⁺ 还原成砖红色沉淀！
• 如何用化学试剂区分醛糖（如葡萄糖）和酮糖（如果糖）？ 不能用碱性的 Fehling 试剂，而要用弱酸性的溴水（Br₂/H_2O）！因为在弱酸条件下酮糖无法发生烯二醇异构，溴水只能把醛糖氧化为醛糖酸（溴水褪色），而完全不能氧化酮糖。
• 临床酶法测血糖与华农 2024 判断真题大坑：注意：千万不要以为“单糖只有打开成直链状才能发生氧化还原反应”！临床上用于精准测定血糖的葡萄糖氧化酶（Glucose oxidase），恰恰是高度专一地直接催化环状 β-D-吡喃葡萄糖氧化为 D-葡糖酸-δ-内酯（或 γ-内酯）和 H_2O2（溶液中 α 型经变旋酶转为 β 型后被一并测出） 2024·判断！此外，王镜岩教材严谨表述为“所有醛糖和许多酮糖是还原糖”，做判断题时提防“所有酮糖在任何条件下都能被温和氧化剂氧化”之类的绝对化陷阱。

3D3D 糖类：单糖立体异构、成环变旋与化学性质交互查看 D/L 构型与差向异构体、Haworth 成环（上D顺β）、变旋现象与 β-D-吡喃葡糖全平伏键椅式构象、还原糖与糖磷酸酯↗

1. 强氧化与部位专一氧化——三类糖酸（Sugar acids）：

• 醛糖酸（Aldonic acid）：仅氧化 C1 醛基（如溴水氧化或葡萄糖氧化酶作用），如D-葡糖酸（临床用葡糖酸钙补钙，戊糖磷酸途径中有 6-磷酸葡糖酸）。
• 糖二酸（Aldaric acid）：用热稀硝酸（HNO₃）强氧化，使 C1 醛基和末端 C6 伯醇基同时氧化成羧基。必考特例：D-半乳糖和 L-半乳糖被硝酸氧化后，由于分子内部出现对称平面，生成的是同一种内消旋的半乳糖二酸（又称黏酸 Mucic acid），完全没有旋光性且难溶于水，常用于鉴定半乳糖！
• 糖醛酸（Uronic acid）：在酶促或保护 C1 半缩醛的条件下，只氧化末端 C6 伯醇基而保留 C1 醛基，如 D-葡糖醛酸（GlcUA，肝脏解毒结合剂及糖胺聚糖构件）、L-艾杜糖醛酸（IdoUA）和 D-半乳糖醛酸（果胶主链构件）。

1. 还原反应——糖醇（Alditols）：
   用硼氢化钠（NaBH₄）或催化加氢将单糖羰基还原为羟基，得到多元糖醇：

• D-葡萄糖还原得到 D-山梨糖醇（Sorbitol，即 D-葡糖醇）；而 D-果糖因为 C2 酮基被还原时会产生新的手性方向，会同时得到 D-山梨糖醇和 D-甘露醇（Mannitol） 两种产物！
• 重要生理与临床糖醇：① D-甘露醇：临床上广泛用作渗透性脱水剂，用于降低颅内压、眼内压及治疗急性肾功能衰竭；② 木糖醇（Xylitol）：甜度与蔗糖相当，代谢初期不依赖胰岛素，防龋齿；③ 核糖醇（Ribitol）：是维生素 B₂（核黄素，FMN/FAD 前体）及革兰氏阳性菌细胞壁磷壁酸的组成成分；④ 肌醇（Inositol，环己六醇）：9 种立体异构体中唯独肌-肌醇（myo-inositol）有生物活性，其磷酸化产物肌醇-1,4,5-三磷酸（IP₃）是关键的胞内第二信使（动员内质网 Ca²⁺），而植物种子中的肌醇六磷酸酯称为植酸（Phytic acid）。

1. 成脎反应（Osazone formation）：
   还原糖在弱酸加热条件下与过量（3 分子）苯肼（Phenylhydrazine）反应，不仅 C1 羰基连上苯肼，连紧邻的 C2 羟基也会被氧化并连上苯肼，生成不溶于水的黄色结晶——糖脎（Osazone）。因为成脎反应同时占据了 C1 和 C2 并消灭了 C2 的手性中心，所以仅在 C1 和 C2 上结构不同的 D-葡萄糖、D-甘露糖（C2 差向）和 D-果糖（C2 酮糖） 与苯肼反应后，会生成完全相同的同一种葡萄糖脎黄色结晶！
2. 三大类重要单糖衍生物：

• 氨基糖（Amino sugars）：单糖的一个羟基（几乎总是 C2-OH）被氨基（-NH₂）取代，且常以 N-乙酰化形式存在，如 N-乙酰-D-葡糖胺（GlcNAc）、N-乙酰-D-半乳糖胺（GalNAc）；细菌细胞壁特有的 N-乙酰胞壁酸（MurNAc） 是由 GlcNAc 的 C3-OH 与 D-乳酸形成醚键而成；而 唾液酸（Sialic acid，即 N-乙酰神经氨酸 Neu5Ac） 是一种 9 碳氨基糖酸，带有负电荷，常位于细胞膜糖蛋白和神经节苷脂寡糖链的最外端，作为细胞识别标志（如老化红细胞丢失唾液酸后即被脾脏巨噬细胞识别清除）。
• 脱氧糖（Deoxy sugars）：单糖羟基被氢原子取代，如 DNA 中的 2-脱氧-D-核糖，以及 6-脱氧己糖 L-鼠李糖和 L-岩藻糖。
• 糖磷酸酯（Sugar phosphates）：如 G-1-P、G-6-P、F-6-P、F-1,6-BP。为什么细胞一旦把葡萄糖吸进来，第一步就要把它变成糖磷酸酯？因为磷酸基团在生理 pH 7.0 下带有近 2 个单位的负电荷，带电的糖磷酸酯无法穿过疏水的脂双层细胞膜，从而将糖分子牢牢锁在细胞内，同时磷酸基还能作为酶活性中心的识别把手并降低活化能 2012·单选！

二、缩醛成苷反应与糖苷键：锁定异头碳的魔法

当环状单糖的半缩醛（或半缩酮）羟基（即异头碳上的 -OH）在酸或酶催化下，与另一个分子（可以是醇、酚、胺，也可以是另一分子糖）的基团缩合脱水时，半缩醛就变成了真正的缩醛（Acetal）或缩酮（Ketal），生成的化合物统称为 糖苷（Glycoside）：环状单糖的半缩醛（或半缩酮）羟基与另一化合物（配基）缩合脱水形成的缩醛（或缩酮）衍生物；提供半缩醛羟基的糖部分称为糖基（Glycosyl），非糖部分称为糖苷配基（Aglycone），二者之间的连接键称为糖苷键（Glycosidic bond）。

根据连接原子的不同，糖苷键可分为 O-糖苷键（最常见，如二糖、多糖及 Ser/Thr 连接的糖蛋白）、N-糖苷键（如核苷酸中核糖 C1 与碱基氮相连，以及 Asn 连接的糖蛋白）、S-糖苷键和 C-糖苷键。此外，自然界中还有许多具有强生理活性的天然糖苷，例如 强心苷中的乌本苷（Ouabain）是细胞膜 Na⁺,K^+-ATP 酶的强效特异抑制剂。

这正是理解糖苷及二糖还原性的核心钥匙：

半缩醛（游离异头 -OH）在水中可以自发开环变回醛基，所以有变旋现象、有还原性、能成脎；而一旦异头碳参与形成了糖苷键，半缩醛就变成了缩醛（Acetal），缩醛在水中和碱中极其稳定（仅能被酸或专一糖苷酶水解），绝不能自发开环！因此，凡是异头碳被糖苷键锁死的简单糖苷，一律没有变旋现象、不能还原 Fehling 试剂、也不能与苯肼成脎！

三、五大核心二糖与寡糖的全景对比：谁有还原端？谁被彻底锁死？

当糖苷配基是另一分子单糖时，就形成了二糖（Disaccharides）。二糖的缩합共有两种截然不同的连接方式：

1. 半缩醛羟基 + 醇羟基（单向箭头 →，保留一个游离异头碳 = 还原性二糖）：
   第一个单糖提供异头碳（C1）上的半缩醛羟基，第二个单糖提供非异头碳（通常是 C4 或 C6）上的普通醇羟基。此时，第一个单糖的异头碳被锁死在糖苷键中，称为非还原端（Non-reducing end）；而第二个单糖的异头碳（C1）依然保留着游离的半缩醛羟基，随时可以在水中开环暴露醛基，称为还原端（Reducing end）！

• 寡糖与多糖的标准书写与命名法则：按国际惯例，非还原端写在左边，有游离异头碳的还原端写在右边；系统命名时必须从非还原端读向还原端，用单向箭头 (α/β 1→ 4) 表示！
• 麦芽糖（Maltose）：淀粉被淀粉酶水解的主要产物，饴糖的主要成分（甜度约为蔗糖的 1/3）。由两分子 D-吡喃葡萄糖通过 α(1→ 4) 糖苷键 相连，简写为 Glc(α1→ 4)Glc（全称：O-α-D-吡喃葡糖基-(1→ 4)-D-吡喃葡糖）。因为右端葡萄糖有游离 C1-OH，故是还原糖，有变旋现象。
• 纤维二糖（Cellobiose）：纤维素不完全水解的产物。同样由两分子 D-吡喃葡萄糖组成，但连接键是 β(1→ 4) 糖苷键，简写为 Glc(β1→ 4)Glc，是还原糖。（注：两分子葡萄糖连接而成的葡二糖共有 11 种异构体，除麦芽糖和纤维二糖外，还有异麦芽糖 Glc(α1→ 6)Glc、龙胆二糖 Glc(β1→ 6)Glc 和海藻糖等）。
• 乳糖（Lactose）：存在于哺乳动物乳汁中（约占 5%）。由左端的 β-D-吡喃半乳糖提供 C1-OH，与右端的 D-吡喃葡萄糖的 C4-OH 通过 β(1→ 4) 糖苷键 相连，简写为 Gal(β1→ 4)Glc（全称：O-β-D-吡喃半乳糖基-(1→ 4)-D-吡喃葡糖） 2026·单选。因为右端葡萄糖保留游离异头碳，故乳糖是还原糖。

1. 半缩醛羟基 + 半缩醛（酮）羟基（双向箭头 ↔，无游离异头碳 = 非还原性二糖）：
   如果两个单糖双双拿出各自的异头碳羟基面对面缩合脱水，那么两个糖环的锁眼（异头碳）就全都锁死在了同一个糖苷键里！整个分子没有任何游离的半缩醛（酮）羟基，哪一头都打不开环！因此，这类二糖没有还原端，属于非还原糖，不能还原 Fehling 试剂，没有变旋现象，也不能成脎！书写时必须用双向箭头 (↔) 标明两个异头碳相连：

• 蔗糖（Sucrose）：日常食糖（白砂糖），仅由光合植物合成而动物体内不存在，是植物体内长距离运输糖类的主要形式。它由一分子 α-D-吡喃葡萄糖（提供异头碳 C1） 与一分子 β-D-呋喃果糖（提供异头碳 C2） 头碰头缩合而成，糖苷键简写为 Glc(α1↔ 2β)Fru（全称：O-α-D-吡喃葡糖基-(1→ 2)-β-D-呋喃果糖苷） 2025·单选。
• 为什么植物选蔗糖作筛管运输糖？ Vander Heiden 教授一语道破：因为蔗糖把葡萄糖的 C1 醛基和果糖的 C2 酮基全部锁死在 (α1↔ 2β) 糖苷键中，使其成为化学性质极稳定的非还原糖，在植物筛管长途运输中绝不会发生非特异氧化或与蛋白质发生副反应，只有到达靶器官被蔗糖酶切开后才释放能量！
• 蔗糖的“转化（Inversion）”与转化糖（Invert sugar）：蔗糖本身是右旋的（比旋光度 +66.5^°）；但当用稀酸或蔗糖酶（Invertase，又称转化酶）将其水解为等摩尔的 D-葡萄糖（+52.6^°）和 D-果糖（-92^°）混合物时，由于果糖的左旋度远大于葡萄糖的右旋度，水解液的比旋光度会由正变负（变为 -19.8^°）！这一旋光方向反转的过程称为蔗糖的“转化”，所得等摩尔葡萄糖与果糖的混合物称为“转化糖”（蜂蜜的主要成分就是天然转化糖）。
• 海藻糖（Trehalose）：广泛分布于藻类、真菌（蘑菇、酵母）、地衣和节肢动物中，是 昆虫血液（血淋巴）中主要的循环能源糖。它由两分子 α-D-吡喃葡萄糖通过各自的 C1 异头碳相连，简写为 Glc(α1↔ 1α)Glc，同样是典型的非还原性二糖。
• 其他重要非还原性寡糖：
• 棉子糖（Raffinose）：植物中的非还原性三糖，结构为 Gal(α1→ 6)Glc(α1↔ 2β)Fru（相当于在蔗糖的葡萄糖 C6 上再接一个 α-半乳糖；用蜜二糖酶水解明得半乳糖+蔗糖，用蔗糖酶水解得果糖+蜜二糖）。
• 环糊精（Cyclodextrins）：由 6、7 或 8 个 D-葡萄糖分子通过 α(1→ 4) 糖苷键首尾相连形成的环状寡糖（分别称 α,β,γ-环糊精）。因为首尾成环消灭了仅有的还原端，环糊精属于非还原糖，且具有独特的“圆筒内部疏水、外部亲水”结构，在医药和食品工业中广泛用作疏水药物的包埋增溶剂、稳定剂和人工模拟酶。

3D3D 糖类：寡糖糖苷键与还原性/非还原性二糖交互对比麦芽糖 α(1→4)、乳糖 Gal(β1→4)Glc、纤维二糖（有游离异头碳还原端）与蔗糖 Glc(α1↔2β)Fru、海藻糖、环糊精（无还原端）↗

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🎬 MIT 7.05 Lec 12 · Carbohydrates & Introduction to Membranes⏱ ~50:00

~00:00MITOCW | 12. Carbohydrates/Introduction to Membranes

~00:02PROFESSOR:

~00:02OK. Great. Now let's dive into the material today. So today's topic is carbohydrates as well as an introduction to membrane structure. And from the very first class, I believe Professor Yaffe talked to you about the four main classes of biological molecules. So proteins and amino acids, you've spent a lot of time talking about that. Nucleic acids is coming up. And the other two are carbohydrates or sugars and lipids. Today, we're really going to focus most of the lecture on carbohydrates, what they are, structure, something about nomenclature. And then at the end, we're going to talk a little bit about lipids and basic membrane structure. Now carbohydrates and lipids are really critical energy storage molecules for cells. And when we talk about metabolism-- the most interesting part of this course-- after spring break, we're going to delve into this in a lot more detail. But sugars are also very important to understand nucleic acid structure. Turns out, membranes are essential for signal transduction. Those are the two major topics that Professor Yaffe is going to talk about for the rest of his time. And so this year, we're going to try something different and have me introduce at least some of these topics now, which will deal with some redundancies that otherwise might have existed and maybe set it up better for him to discuss some of the lectures coming up. Now today's lecture is unfortunately topically a little bit disjointed. But it still has important information about biochemistry. And it will help us all speak the same language, both for the upcoming lectures from Professor Yaffe as well as things that I will start off with when I come back. And it's a nice way to ease back in after the exam. OK. So what is a carbohydrate or a sugar? So let's break this down, and so it's carbohydrate. So a carbohydrate is effectively carbon in some ratio with water. So all carbohydrates have the same chemical formula, Cn and H2On. And so there are some deviations from this in biology. Sometimes, you can introduce a heteroatom, phosphate, sulfur, nitrogen, et cetera. Technically, these are not carbohydrates, although they're often lumped together with carbohydrates. And why would nature do this? Because it changes some of the chemical properties that can be useful for either structural or signaling reasons. You may encounter these later-- certainly, in other classes. But we're not going to talk much more about that today. We really just kind of focus on the base carbohydrate, Cn H2On structure. Now carbohydrates come in different forms. And so they can come as single units with that structure. These are so-called monosaccharides. Or these single units can come together to form various polymers. And those polymers could be two units, so-called disaccarides-- so two sugars stuck together. Or many units, and so sometimes, those are referred to as oligosaccharides or polysaccharides. And there's really no clear distinction between a few chains together, oligosaccharides, many polysaccharides. They're somewhat used interchangeably.

~03:28Now you guys have almost certainly heard of many of these things. So what's a monosaccharide? So a good example of that is glucose. So glucose is, of course, the main sugar that exists in your blood. What's a disaccharide? So a common one is sucrose. So sucrose is a disaccharide-- so two sugars stuck together, glucose plus fructose. You've probably heard of both of those things before. Sucrose is, of course, table sugar. It's what you would have mixed into your coffee if you had that this morning. And an example of a polysaccharide is starch-- so what's in a potato. All right. Now clearly, these are all sugars. They're all relevant to a human diet. You would never confuse-- you've probably not knowingly tasted glucose. It's not particularly sweet. Sucrose is much sweeter. And a potato is not necessarily sweet at all. Yet, this really points out that how these sugars are built, the different structures matter a lot. They matter for things like how you taste them in your diet. And how carbohydrates are built also matters a lot for all kinds of aspects of biology, which is why it's somewhat important to at least understand some of what we're talking about-- a common language about how to describe these molecules. OK. So the simplest biological sugars have three carbons that are least commonly used. These are referred to as trioses. And if we take the general formula, C3 H2O3, and we say, what are two ways that we can satisfy that formula? There's two main ways we can do it. One is like this. So if you add up-- count all the carbons, hydrogens, and oxygens, you will see that that is C3 H2O3. This molecule is called glyceraldehyde. OK. And the other way we can do this is like this. OK. Again, three carbons, three waters. If you add up all the atoms, this molecule is called dihydroxyacetone. And these are sugar-- same chemical formula, different chemical structure. That has a term. That's called an isomer. And it turns out that we can chemically interconvert these molecules in the following way. And so if we carry out this chemistry, we will get this intermediate.

~05:58OK. And that will allow you to interconvert glyceraldehyde-- this aldehyde-- with this ketone, dihydroxyacetone. And the enzyme class that carries this out is a class of enzymes called isomerases. And this is exactly the chemistry that that enzyme would use to interconvert these two forms of this triose, this three-carbon sugar molecule. All right. Now if we look at dihydroxyacetone, there's no stereocenter here. What do I mean by a stereocenter? That's a carbon that has-- reminder from 512-- carbon that has four different non-equivalent substituents around it. However, if you look at glyceraldehyde, that carbon in the middle is a stereocenter-- four non-equivalent groups around it. And so there's two ways that I can draw glyceraldehyde. I can draw it like this. Or I could draw it like this. All right-- so two different ways. So the one on the left here is D-glyceraldehyde. And the one on the right is L-glyceraldehyde. OK. And I know that this is review. Some people are good at seeing these things. Some people are not. I brought a couple of models here. Here's this-- the blue and the brown are two stereocenters with four different constituents on them. No way you can twist these around to make them identical molecules. Why does this matter? Well, enzyme-active sites are going to fit this molecule different than this molecule. And this is why these stereoisomers matter so much for biology. It's also something that's really hard to accomplish.

~07:38If you think about how do you actually generate stereoisomers if you were in an organic chemistry lab-- really hard. But biology does this all the time. And the real reason is because it's enzymes that ultimately catalyze these interconversions. And different stereoisomers will fit differently into enzyme-active sites. Now the way that I've been drawing these sugars is a convention called a Fischer projection. And when drawn in this way, the convention is that-- so you put the carbonyl towards the top. If the OH group, the alcohol, is pointing to the right, that's D. If it's pointing to the left, that's L-- and so D, pointing to the right, L, pointing to the left. Like amino acids, biology has chosen one stereochemistry for most biological sugars because, of course, enzymes act on them. And so in biology, it's D, sugars. This is in contrast to L, amino acids. And so if you can remember that sugars are D, you know that amino acids are the opposite. Or if you remember that amino acids are L, you can remember that sugars are the opposite. OK. All right. So dihydroxyacetone is really the only sugar with three or more carbons that doesn't have a chiral center. Everything else will. And so if I go to a four-carbon sugar-- draw a couple of them here. OK. So here's C4 H2O4 drawn with a ketone. If you look at this, this is a chiral center. OH group is pointing to the right. This is a D sugar. If I had drawn it with the OH group on this side, it would be an L sugar. All right. If I draw this sugar as a different isomer, this time with an aldehyde, now, we encounter a bit of an issue because now I have one, two stereocenters. OK. So if there's two stereocenters, that means there's 2 to the n ways that I can draw this as a stereoisomer. And so you can see that this could get really complicated very quickly. Now this sugar obviously is a D because I drew both OH groups pointing to the right. But you can imagine, I could draw one this way or one that way. And so how do if it's a D or an L sugar? And so the convention is that whether or not a sugar is designated as D or L refers to the stereocenter that is furthest away from the carbonyl. And so this is the relevant stereocenter that says it's a D sugar. So what do I mean? I can draw. And then any other sugar would have a different name. And so what do I mean by that? Let me draw this a few different ways. And so the sugar that I've drawn here is referred to as D-erythrose. All right. If I draw it, now the OH group on the carbon furthest from the carbonyl pointing to the left, so this would be L-erythrose. And if I draw it differently by altering the stereochemistry of this carbon, now, it has a different name. And so this carbon is D-- or this sugar is D-threose. All right. Makes sense. If I flip the OH group to this side, it would be L-threose. All right. So lots of possibilities. Turns out nature only uses a subset of the stereoisomers and makes them relevant for biology. For example, Derythrose is something that you will encounter when we talk about metabolism later in the course. D-threose, as far as I know, is not used in biology. I would never say, it's never used in biology. Never bet against biology. It can do absolutely everything. There's always an exception somewhere. But, in general, D-threose is not something that really exists, at least commonly in nature. All right. So if we go through, and you look at all of these different sugars that I've drawn, you can see that they either have an aldehyde, or they have a ketone somewhere in the molecule. All right. So if you have an aldehyde, these sugars are generically referred to as aldoses. All right. And if you have a ketone, an internal carbonyl, these are generically referred to as ketoses.

~12:24Now you might say, as you start getting to longer and longer sugars, you can put the ketone anywhere along the sugar, and there would be a ton of different possibilities. But it turns out ketoses always have the carbonyl as the second carbon in from the end. The reason for that is because biology, as you will see when we talk about metabolism, interconverts these via isomerase reactions. And so you can't use an isomerase reaction to interconvert a ketose and an aldose unless the ketone is one carbon away from the end of the sugar. And so this fact really limits some of the diversity of ketoses that can actually exist in nature. Now most important biological sugars, at least the most common ones, end up having six carbons or five carbons. And these are referred to as hexoses or aldose-- or pentoses. Sorry-- so much nomenclature. OK. So sixcarbon sugars are hexoses. Five-carbon sugars are pentoses. And if we just talk about the hexoses, you're very familiar with a couple of them. And so one that we mentioned earlier, blood sugar-- glucose looks like this. So this molecule is D-glucose. It's a D sugar because the stereocenter here furthest from the carbonyl-- OH group points to the right. So it's a D sugar. It's an aldose because it has an aldehyde. All right. And it's a hexose because it has 1, 2, 3, 4, 5, 6 carbons. And what makes it glucose is the stereochemistry of these other sites that end up being-- this is the molecule glucose. Now if I carry out that isomerase reaction that I showed you earlier, it'll give me this intermediate. Let me draw the whole thing. Then I interconvert that aldose. Now, it becomes a ketose. This is D-fructose-- another common sugar that certainly is in the news a lot. D sugar because the stereocenter for this from the carbonyl points to the right, makes it a D sugar. It's a ketose. It's a hexose. This organization of other stereocenters is what makes it D-fructose. All right. Now if we go through and we count these, there's four stereocenters in glucose, three stereocenters in fructose. That means 2 to the n. There are 16 ways I can make ketose aldose, eight ways I can make a ketose-- I'm sorry-an aldose hexose, eight ways I can make a ketose hexose. Among those, that's even 12 different ways that I can have D hexoses that are a ketose or an aldose. Could be very, very complicated. But it turns out, fructose is the only D hexose ketose that really is relevant for nature. There's only two other molecules related to glucose-- only two other hexose aldoses that are sugars used in nature. You've probably heard of these as well. I'll draw them to illustrate a different point. OK. So this molecule is galactose, an important sugar found in milk. All right. If I draw a glucose next to it again, glucose-- so these two sugars differ here, the stereochemistry there. So they are, I guess, isomers of each other. There's a special name for it I will get to in a second. So that's how galactose is related to glucose. The other major aldose hexose is this one. This is D-mannose. This differs from glucose here-- that carbon at that carbon. By the way, by convention, the way that you number carbon in sugars is you start with the end that's closest to the carbonyl, either the aldehyde or the carbon one up from the ketone, so this would be carbon 1, 2, 3, 4, 5, 6. So galactose differs from glucose at carbon 4. Glucose differs from mannose at carbon 2.

~16:36These isomers have names in relation to each other. And so two sugars that differ by one part of stereochemistry-- so galactose to glucose or glucose to mannose-- are called epimers. And these can be or converted by enzymes called epimerases. We'll talk about how these work later in the course. And so glucose is an epimer of galactose. Glucose is an epimer of mannose. Mannose is not an epimer of galactose because mannose and galactose differ at both carbon 2 and carbon 4-- their stereochemistry. Why is that relevant? Because if you're going to interconvert galactose and mannose, you would have to do it in two steps, two different epimerase reactions to interconvert those two sugars. All right. Great. So we've discussed now all of the major hexoses that nature uses. We've discussed all the major trioses that nature uses. The other major length of sugars that ends up being important in biochemistry is the five-carbon sugars, the pentoses. And so I want to mention a couple pentoses and do so in a way that will allow me to basically solidify some of the nomenclature that I've gone through. And, of course, one of the five carbon sugars-- ribose-- is really critical to nucleic acid structure, which is one of the reasons why we're talking about this at this point in the course. OK. So this is a pentose-- 1, 2, 3, 4, 5-carbon sugar, D-ribose. All right. It's a D sugar because the stereocenter furthest from the carbonyl points to the right. It's an aldose because it has an aldehyde group. I can act on this with an isomerase. I'm not going to draw out the isomerase reaction again. It's exactly what I drew before. If I did this, now, I turn this into a ketose. Pentose because it's five carbons. Ketose because as a ketone. D sugar because the stereocenter furthest from the carbonyl points to the right. This has a name of D-ribulose. All right. And it turns out that there is an important epimer of D-ribulose that's found in nature. The epimer changes the stereochemistry at carbon 3-- carbon 1, 2, 3, 4, 5. That's carbon 3. And so if this was acted on by an epimerase that did that, you get this sugar, also a pentose, also a ketose, a D sugar, but an epimer of ribulose. It's called D-xyulose. It's an epimer because xyulose ribulose are epimers because they differ by stereochemistry only at one position. I'll just say, right off the bat, you should not memorize names of sugars and their structures. These are things you can look up in books. The point of going through all this is just to expose you to some of the nomenclature, remind you about stereochemistry. I realize, these are basic things. Many of you have already encountered this. Some of you find this very easy. Some people find these sort of spatial things more difficult. This is very well-reviewed, though, in textbooks or other places online if you need to look it up. But the key thing is just to remember this nomenclature because it'll make it easier for us to talk about sugars later in the course. All right let's take a short break, so I can get some board space back. And then we'll build off some of these concepts in a minute. I've been drawing all of these sugars as straight chains. But you probably know, from high school or from looking at DNA or RNA that the ribose there is not a straight chain but, instead, forms a ring. And, in fact in solution, particular aqueous solution, sugars, particularly five carbons and longer almost always exist as rings. And there is a very clear reason for this. And you probably remember, from 512 Organic Chemistry that alcohols will react with aldehydes and ketones in solution.

~20:58And so here, I have a model of glucose and fructose if you want to come and play with them. And so if you look at this, and you just look at the model, you see that this oxygen, right here, this alcohol, in space, is very close or can be moved to be very close to this aldehyde here on the end of the molecule. Or the same thing here with fructose. Here's a alcohol very close in space with this ketone. All right. So what happens in this situation? Well, if you have-- this is a review of organic chemistry. OK. So here's any generic aldehyde. Here's some alcohol. OK. So those things react. You end up with this so-called hemiacetol. Or the same thing if I do it with a ketone and an alcohol-- now you get this hemiketal. OK. Now given that you have an alcohol reacting with a carbonyl, an aldehyde, or ketone on the same molecule, well, what happens is you effectively get a ring with oxygen being one of the components of the ring. And so if we draw this for glucose-- so this is D-glucose. All right. And so if the alcohol here on carbon 1, 2, 3, 4, 5 interacts with the aldehyde on carbon 1-- you can play with the model and see that that is the one that's close in space-- you now end up getting this. This, where you get a ring between carbons 1 and 5. We can number the rest of them-- 2, 3, 4, 5, 6. All right. Or if I turn this molecule so that you can now draw it in a slightly more chemically proper way, now, you basically have-- this is carbon 1, 2, 3, 4, 5, 6. So carbon 5, oxygen from carbon 5 now bound to carbon 1 gives you this six-membered ring structure. The six-membered ring structure is reminiscent of an organic molecule called a-- looks like that-- called pyran. And so this six-membered ring in a sugar is also referred to as a pyranose. All right. So that's the first thing. The second thing is that this whole business can be very tedious to draw. In fact, it's very tedious drawing sugars in general, as I'm sure you would agree with me if you're taking notes during this lecture. And so oftentimes, these pyranoses, like glucose, are drawn with shorthand. And the shorthand is as follows, where I basically represent the OH groups as simply lines. And so this is another shorthand to draw that pyranose form of glucose-again, carbon 1, 2, 3, 4, 5, 6. Now the last thing is you can see I didn't draw the OH group there on carbon 1. And that's because by making this pyranose ring, if you look at carbon 1, I have now generated a new stereocenter. And so carbon 1 now has four non-equivalent groups on it, which means I could draw the OH group, that carbon 1, in two different ways. And so this is carbon 1. I could draw it such that the OH group points down. Or I could draw it such that the OH group points up. And those are two different molecules. And so there's a naming convention for this too. And so if the OH group points down using this way of drawing the molecule, it's called alpha. If the OH group points up, it's called beta. All right. And so this alpha versus beta ends up being structurally different because it puts, basically, that OH group pointing in a very different position in space. So if I make a ring here with glucose, and the OH group is pointing here versus there-- very different position in space. And this has implications for how you build bonds for disaccharides and polysaccharides that make structural differences. And we'll cover this a lot when we get to metabolism.

~25:24But it should be very clear now that glucose, if you just take a solution of glucose and put it in water, it is not one thing. There's actually multiple different forms it can have. It could be as I drew it up here with the OH group pointing down. This would be alpha D-gluco-- because it's glucose-- pyranose, because it's in the pyranose ring form. I could have drawn it with the OH group pointing up-- different molecule. That would be Beta Dglucopyranose. Or it could just be the open-chain D-glucose that I was drawing earlier. All right. All of those are perfectly legitimate ways for glucose to exist in solution. All right. Now it turns out, that in reality, about a third of it in solution is this. Two thirds is that. And a trace amount is this. OK. And that has to do with just what is more favorable forms or not. But which form it's in actually matters for structural reasons, as we'll see later in the course. Now the final complexity is that this ring is not flat. So if I actually take glucose here and make a form of it-- so here's my glucose molecule. There's no way for me to make this completely flat, say, like benzene. And so there's really two different pyranose confirmations that can be formed. I'll try to draw them, but they're harder to draw. Here's a overhead that you can look at if it's easier. But basically, you can have it form this so-called boat form or this so-called chair form. So there's the boat versus the chair conformation. Glucose, turns out, prefers the chair conformation. And there's an interesting thing that comes from this because if you take beta D-glucopyranose, it turns out of all the hexoses that exist in possible hexoses that exist, the form of a hexose aldose that best spreads out all the hydroxyl groups is beta D-glucopyranose, the more common one in solution. Why does this matter? Well, because if this has this reactive aldehyde bound up in this stable ring structure, it's less likely to react with other aldehydes in the cell. And so this is likely why nature chose D-glucose as the most common storage sugar-- why it's sugar in your blood-- because it's the most stable hexose that's out there. And it's not just a random reason that nature picked this one, but actually because of real chemical stability issues for why it's there. All right. Now I want to mention that ketoses also can form rings. And I'm going to use this as an example to show you that a ketose-- so here's fructose. So this is D-fructose. And so it turns out, this can form two possible rings. It can form a five-membered ring or six-membered ring. So how do I form a five-membered ring? So if I take the carbon here from carbon 1, 2, 3, 4, 5, the hydroxyl from carbon 5, form a ring there. Now, I get this molecule. So there's 1, 2, 3, 4, 5, 6. OK. Or if I now turn this so that I draw it in the way you're probably more used to seeing it, I'm going to use the shorthand here. So this here would be carbon 1, 2, 3, 4, 5, 6-- hydroxyl from carbon 5, forming a bond to carbon 2. That creates a new stereocenter at carbon 2. OH group is pointing up. So this is a beta sugar-- OH group pointing up. So this here, as I drew it, would be beta D-fructo and this is a furanose. Why is that? Because the organic molecule that's a five-membered ring with an oxygen in it is a furan. And so the five-membered ring is referred to as a furanose-- beta D-fructofuranose. If I'd drawn it with the OH group pointing down, it would be alpha Dfructofuranose. OK. There's another possible ring I can do. And instead, I take the hydroxyl from carbon 6 and do that-- form a ring. Now I'm going to form a six-membered ring. We have carbon 1, 2, 3, 4, 5, 6.

~30:05So if I now turn this-- so this, here, now I drew the OH group pointing down. So it's an alpha. If I drew it up, it would be beta. So this is alpha D-fructofuranose-- six-membered ring version of fructose, five-membered ring version of fructose. So lots of non-equivalent ways I can draw fructose. And it turns out, these actually matter. And they matter for real things that probably matter to you. And so I brought with me here two different sweeteners. This is corn syrup. This is honey. Has anyone ever-- I'm sure most of you have had honey. Has anyone ever tasted corn syrup? Come on. Someone's tasted corn syrup. Is a sweet? Which one's sweeter? Honey, by far-- much, much, much sweeter. I used to have it so you guys could come up and taste them. But I couldn't come up with a way to do that in a sanitary way. So I gave up on it. But nonetheless-- much sweeter than that. It turns out, these are not pure fructose. They're a combination of sugars. But their composition is actually, from a chemical standpoint, similar amounts of fructose in each one. And it turns out that honey is beta D-fructopyranose. Whereas corn syrup is beta D-fructofuranose. All right. So same sugar, different structure-- one's a furanose, one's a pyranose-- tastes very differently to you, one being much more sweet, one being much less sweet. OK. Now, of course, I also want to talk about ribose because it's the thing that you guys are going to talk about the most next because it's in nucleic acids. And so just as a reminder, here's ribose. It's a aldose and a pentose. So this is D-ribose. Ribose, as from high school, forms five-membered rings. That's because it links alcohol on carbon 4 to the aldehyde on carbon 1. And that gives you this ring structure. Now number of carbons-- 1, 2, 3, 4-- alcohol on carbon 4, forming a ring to the aldehyde on carbon 1. This is carbon 5. And the way I drew it, OH group pointing up-- so this is beta D-ribofuranose would be the proper way to have it. And so when you guys talk about this in DNA, well, the base is going to be linked to carbon 1. It's going to replace that hydroxyl group-- the beta form of the hydroxyl group with the nitrogen. And you're going to talk about bonds being from the 5 prime or the 3 prime end. Those are those hydroxyl groups. That's where 5 prime and 3 prime comes from because those are the 5 and the 3 position on ribose. Great. Perfect. All right. So we will return to carbohydrates in great detail. We'll discuss how we combine different carbohydrates, different monosaccharides to make disaccharides and polysaccharides, how these different structural properties end up mattering for things like energy storage as well as to produce various structural molecules that can be important for different cells in organisms. But for the remaining part of the class today, I really want to shift topics to now discuss a completely different class of biomolecules. And that's lipids. So as I said earlier, the reason we're going to do this is because coming up for Professor Yaffe's lectures, lipids end up being really important molecules for various aspects of cell signaling. They're also very important for energy transduction, which is why I'm talking about them as well. We will spend a lot of time later talking about lipids in great detail. But really, what I want to focus on today is how lipids are used to create membranes. That is, barriers that really separate the outside world from the inside of cells, things that make compartments within cells, and these things also end up being surfaces where you can have key things happen. So you reduce spatial complexity if you go to a 2D surface versus a 3D surface, which is part of why they're so important in signal transduction.

~34:38But to talk about these, I have to introduce, first of all, what is a lipid? So lipids are a class of molecules. You think of them as fats. That's how they fit in the nutritional standpoint of what we'll all talk about. But first, I want to give a general definition of what a lipid is. Now there's lots of different classes of lipids. And we'll cover these later in the course. But in general, at the highest level, almost all lipids consist of two pieces-- consist of something called a fatty acid that's esterified to an alcohol. So what is a fatty acid? Well, a fatty acid is really any molecule that has a carboxylic acid group. So there's a carboxylic acid. And then hooked up to that carboxylic acid is a whole bunch of saturated hydrocarbons- alkyl chains. Oftentimes, fatty acid would be drawn like this because of all the different saturated chains. So acid group on one end, really greasy alkyl chain on the other. And it turns out that this can be esterified to an alcohol. So here's just a generic alcohol. And as you certainly remember from organic chemistry, I can use an alcohol and an acid to form an ester. And so then that would be-- so now, I have, basically, this ester bond. And this is esterification between some lipid species and a fatty acid, in general, is the general thing that leads to a class of molecules known as lipids.

~36:19Now why this is particularly useful for biology is that this long, greasy alkyl chain is not water soluble. So if you take oil from your cabinet-- canola oil, or olive oil, or whatever, and you pour it in water, what happens? It doesn't mix together. You get these globs of oil floating in the water. And so if you want to form a barrier between two aqueous compartments, a way to do this is to have, basically, a hydrophobic layer-- a membrane-- basically separate the two aqueous compartments, the inside and the outside of the cell. Now if I take a lipid, and the lipid, I form this ester, and it doesn't have a charged group anywhere on the molecule, this is sometimes referred to as a neutral lipid. So what do I mean by neutral lipid? It's basically taking this, which is intrinsically a fatty acid, is intrinsically a polar molecule. It has this carboxylic acid on the end of it. But if I form an ester linkage to an alcohol, and the alcohol has no charge on it, now, it's just this really greasy molecule. And it turns out, that's exactly what is in your olive oil or in your canola oil in your cabinet. It's basically a bunch of fatty acids-- long alkyl chains esterified to non-charged alcohols, which makes this neutral lipid, this greasy molecule, which is great for energy storage in plants, something we'll talk about. But if you want to mix it together with water, it doesn't do very well. So if you go to make salad dressing, and you pour your oil in with your vinegar, the aqueous thing, you get a bunch of droplets. You don't get a solution. And if you think about it, that's not very good at generating an interface between two compartments. You just get a bunch of droplets. You actually don't get an interface between two compartments. So if you want to make an interface, you have to do something different. You need to have a charge group, like on this fatty acid, that's going to be happy sticking towards the aqueous side and then a hydrophobic portion that's going to be happy sticking to form a barrier. Effectively, that's what soap is. So if you want to wash your hands and use canola oil, it doesn't work very well, right? But what happens if you have a bunch of greasy stuff in the water, and you take a drop of dawn dish detergent and drop it in the water?

~39:12You get this immediate barrier that forms across the top as you get this film where you basically align up all these charged hydrophilic parts to the aqueous pieces and all of the greasy parts to the hydrophobic side. So you end up having this nice charged molecule with this long hydrophobic part. So you have a hydrophilic end and a hydrophobic end. And that ends up being very useful as a way to form an aqueous hydrophobic interface. And this is exactly how soap works, as I'm sure you've learned in other classes. As an aside, you know how you make soap? You basically boil the neutral lipids in lye. So boiling it in base, if you remember from organic chemistry, will break the ester linkage. And now, you have these molecules. And that's how you make soap. All right. Now the way biology does this is they actually assemble lipids that effectively have that same property, where, basically, they have a head group from the alcohol that's charged with a hydrophobic group contributed from these fatty acids. And this is what allows things to assemble into membranes. And so membranes are largely made up of-- and certainly, for the purposes of the upcoming lectures-- so-called phospholipids. So what is a phospholipid? Well, on a phospholipid, the alcohol part of the lipid is derived from a molecule called glycerol. So glycerol looks like this. OK. So this is glycerol. All right. 3-carbon molecule-- three alcohols-- actually, very similar. If you look back in your notes to dihydroxyacetone, the difference between glycerol and dihydroxyacetone is carbon 2 was an alcohol here before it was a ketone. Turns out, that's where glycerol comes from-- dihydroxyacetone getting turned into glycerol. And basically, what a phospholipid is that you esterify a fatty acid to two of the alcohols and another phosphate and charged alcohol to the other hydroxyl group. So what do I mean by that?

~41:24So like this-- so here's ester linkage number one. I'll draw it this way just for ease. This is the carbon 2. Here's ester linkage number two. So that's a fatty acid esterified to 2 and 3. And then you now make an ester to a phosphate. So there's a phosphate at carbon 1. And then you make another ester linkage to another alcohol where R equals alcohol. And that alcohol also turns out to be charged. And so the most common phospholipid membrane lipid is a molecule called phosphatidylcholine, which is that structure with the R group being this alcohol. And so this is choline. OK and so phosphatidylcholine is basically that structure. OK. So this is phosphatidylcholine drawn out in all of its glory. So you have this hydrophilic end. Here, you have a hydrophobic end. That's good. This can point towards the aqueous side. This can point towards the greasy side and make a nice interface. And so another common word for phosphatidylcholine-- an old word for it-- is a molecule called lecithin. Anyone read the labels on the side of your food ever? In fact, there's an old commercial that makes fun of this. Soy lecithin? What's that? I don't want that in my ice cream. Well, what is lecithin? It's phosphatidylcholine. It's basically, why is this done? Well, it's commonly added to ice cream because what is ice cream? And it's an emulsion between fat, the cream, and sugar, which is pretty aqueous solubles, you might guess, from looking at what we drew earlier today. And so by putting phosphatidylcholine in your ice cream, you basically stabilize this emulsion between the aqueous and the fat face. So this is why it's added to lots of food. And in fact, it says, soy lecithin, an emulsifier. That's why it's added.

~43:27You can do this yourself. This is a common trick that many cooks know. So if you make salad dressing, you put a little mayonnaise in your salad dressing. Why do you do that? Well, mayonnaise has eggs in it. Eggs have a lot of phosphatidylcholine. That mayonnaise that you add into your salad dressing basically stabilizes that emulsion between the oil in the vinegar and helps it be more stable and distribute better across your lettuce. I think my favorite example of this is, who's made chocolate chip cookies? Everyone's done this, right? OK. So how do you make chocolate chip cookies? So you take butter and sugar, all right, fat, and something that's very non-- very polar, aqueous soluble sugar. And you try to mix them together. You have to cream it, right? And that takes forever. It's a pain in the butt. It doesn't come together very well. And so you get lazy, and you put the egg in there. And then it all comes together really, really easily. So why is that? The egg is the emulsifier that actually brings it together. Whereas trying to get the butter and sugar to make your cookies nice and fluffy is a lot harder because you're really trying to get these two things to come together. And it's basically this exact chemistry that's taking place when you're doing that act of cooking. All right. Now phosphatidylcholine is not the only phospholipid found in membranes. But I'll just quickly mention what a couple of the other ones are. So it turns out you have two other major phospholipids by abundance and then another phospholipid that's really important for signaling. So I'll list them here first. So there's phosphatidylserine, phosphatidylethanolamine These, it turns out, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine is what makes up the majority of the phospholipids in cell membranes. And then there is a much more minor phospholipid that's important for signaling, which is phosphatidylinositol. So what are these? So serine, ethanolamine, and inositol are just, like choline, different alcohols that can be esterified to the phosphate in exactly the same way I did for phosphatidylcholine. And so what's phosphatidylserine? So if you remember, this is-- so this here is the amino acid serine. I'm sure you remember that. If we esterify that alcohol to the phosphate, there's phosphatidylserine. What's ethanolamine look like? So ethanolamine is this alcohol. So that's ethanolamine. Esterify the alcohol to phosphate. Esterify that phosphate to glycerol-- phosphatidylethanolamine. And the last one is inositol. So what is inositol? Well, inositol is a six-membered hydrocarbon rain where all of the carbons have an alcohol on them. And so if I esterify one of these alcohols on inositol, versus a-- if I esterify one of those alcohols to a phosphate to make phosphatidylinositol, turns out that that ends up being a really useful lipid to make for signaling. And you'll hear a lot about this in a couple of contexts, I believe, from Professor Yaffe later in the course. All right. So this having these general membrane structure or this general phospholipid structure, where you have this hydrophobic part of the molecule and the hydrophilic part of the molecule is really what allows these lipids to come together to form these membrane bilayers that are often drawn like this. And so when I'm drawing it like this, basically, what those two wavy lines represent-- those are the hydrophobic fatty acid parts, and this is the hydrophilic so-called head group.

~47:24That's the charged alcohol stuck on to the end of the-- to the phosphate on the glycerol. And these, of course, assemble such that all of the hydrophilic head groups face an aqueous compartment on either side. OK. And then you have this nice hydrophobic portion that is a membrane. And that is what can create a barrier between two different compartments in cells. Now just to put this into context for the protein structure stuff that you learned about-- so when you learn about all the different ways you have protein structure, of course, you have amino acids that are hydrophilic, amino acids that are hydrophobic. If you have a protein that's floating in solution, it has all the hydrophilic parts on the outside and the hydrophobic parts in the middle. But you can imagine that you can also have proteins that assemble to sit such that they span the membrane-- so hydrophobic parts that interact with the hydrophobic part, the inner part of the membrane-- hydrophilic parts on either side. These can form channels. These can form all kinds of ways to move stuff across membranes. Or you might think that you might have a protein that does something like that-- hydrophobic part on one end, hydrophilic part on the other, and really floats on the surface of a membrane. And you will see that the way cell signaling works is that you basically have lots of protein complexes that will assemble that membranes-- two dimensional space. Those membranes, as well as some of the membrane lipids, can then act as messengers that allow cells to carry out various signals. And I will leave that to Professor Yaffe to talk about. All right. So I will also come back and discuss membranes in a very different context in the context of metabolism during the second half of the course because it turns out, a lot of energy transduction and the way cells really store energy also ends up being really important with respect to membranes. All right. You guys are lucky. You get out a little bit early today. This is the first time that we've done the lectures this way, so I don't have my timing quite right. But I will see you guys again for metabolism after spring break.

🎬 MIT 7.05 Lec 19 · Intro to Metabolism, Polysaccharides & Bioenergetics⏱ ~50:00

~00:00MITOCW | 19. Introduction Metabolism/Polysaccharides/Bioenergetics/Intro Pathways

~00:01MATTHEW

~00:02OK. So welcome back, everybody, to 705. I know this has been a really challenging semester for all of us. I want

~00:09VANDER

~00:09to particularly acknowledge for the seniors out there the tragedy of missing the end of your senior year.

~00:15HEIDEN:

~00:15Hopefully us, MIT can somehow make it up to you. I can say for my part, we will do our best to teach you biochemistry remotely. And just as a couple logistics, a reminder, I'm Matt Vander Heiden. I'm going to be covering the remaining part of 705. For the rest of the semester, we get to focus on biochemistry of metabolism. We're going to learn what metabolism is, why it's important. And basically, this can be quite a polarizing topic. In my experience, people tend to really love or really hate metabolism. It's interesting, because there are some people out there who pretend to hate it, but actually deep down really love it. Hopefully I will inspire you over the rest of the semester to at least have some appreciation for why metabolism is important. And regardless, for those of you who want to go to med school, this is a favorite topic of MCAT Exams. And also you will see that it will come up in many different areas of biology, regardless of whether you go to med school or not. So if you do anything further in biology, hopefully you'll find this material somewhat useful. OK, so getting into what metabolism is. So I think it's good to start with a textbook definition of metabolism. And so metabolism is the chemical reactions that all cells and organisms use to do two things. One, extract energy from the environment. And two, synthesize the macromolecules that make up all life. So really, what metabolism is then is the chemistry that makes life possible. And so understanding metabolism is really something that will help you better understand your food. There's lots of material out there in the popular press about what's healthy and what's not. At least this will allow you to form some of your own opinions about those claims, and how they relate to what really goes on in our cells and bodies. There's lots of implications for medicine, very relevant to agriculture, the energy sector, biofuels come from. And so metabolism really matters to tackle many of the challenges that face us in society. Now as a topic, this can be very daunting. As shown here on this image, we have hundreds, this is a typical metabolic pathway chart that is hanging in the walls of my office, many other laboratories and academic places around the world. If you look at this chart, it's filled with hundreds of enzymes, complex pathways. Our goal here is not to memorize this chart. You can always look up details of any pathway or reaction that you want. The goal here really is to see beyond this complexity. I want you to appreciate why metabolism works the way it does, why this chart is organized as it is. Because what we will find is that this chart is really variations on relatively few reactions. It basically is life repurposing similar chemistry to do many different things.

~02:53I think there is a beauty in how life can use these reactions to get such a diversity of macromolecules, and enable cells to solve all kinds of different problems. You will see that all of the pathways really follow common principles. These are shared across all species and all forms of life. And so at the chemical level, all life is really related. And this is why it's so relevant to understanding many of the challenges in medicine, understanding ecology, evolution. And for you MIT students, you'll see it's also very critical for using engineering approaches based on biology to solve various problems in the world. OK. So now let's start getting into this a bit more. Now, the first topic that we're going to cover over the next several lectures really relates to coming back to sugars and carbohydrates, which I talked about and introduced in my previous lecture. And we're going to do this because sugars and carbohydrates are key energy transduction molecules and cells. And it really facilitates a discussion of the principles for how metabolism works. Now our goal here isn't to memorize pathways. We could read about the glycolytic pathway in an hour. I could draw it up here in a very short period of time. Many of you have probably already done this in high school. Our goal is instead to understand this pathway at a deeper level, really see why the breakdown of sugars works the way it does, how cells use this to release energy, and how that can be used to support other cell functions. And so before we get to that, I want to start by introducing just a couple high-level concepts about metabolism. And so really, based on our definition before, metabolism is really about two things. It's about making stuff, and we have a fancy term for that. That is called anabolism. And it's about breaking stuff down. And the term for that is called catabolism. And anabolism, building stuff-- that is, producing biomass, growing all of the material that's out there, anabolic steroids help you grow. This is anabolism to build stuff. That is one side of metabolism. The other side is breaking stuff down, catabolism. That is, breaking down our food, eating food. Digesting our food is a way to get energy. Now of course, to reproduce, life has to build more cells. You need more stuff. You can't create something from nothing, and so that requires energy input. Anabolism typically requires energy input. Whereas that energy has to come from somewhere. And that's where catabolism comes from. We have to break down food. And ultimately, catabolism is the source for biological energy for many different systems. Now, some of this is actually somewhat intuitive. We all learned as little kids that we need to eat food if we're going to grow up and be big and strong. You also know that if you're going to run a race, you've got to eat a bunch of food, you need that energy to fuel your activity. But maybe what's less intuitive to many of you is we also need energy to sustain life, even if we're doing absolutely nothing. OK, adults, sit on the couch all day, hopefully they're not growing. But they'll still starve if they don't constantly eat some food, even if they're inactive. And so why is that the case? Well, hopefully some of you recall the second law of thermodynamics. So what is the second law of thermodynamics? It's, in effect, the entropy of the universe is increasing. That the universe continually tends towards disorder.

~06:05Life is exactly the opposite of this. Life is actually maintaining extreme order in the face of the second law. And so all life must constantly battle entropy. And many have described life at the very highest level as being really the ability to maintain order, to fight entropy, which of course requires constant energy input. And metabolism is the process that all cells use to do this. Now for humans, I think we all know we need to eat and breathe in order to stay alive. For you future MDs out there, what happens if your heart stops, or you stop breathing? Well, of course, you die in a matter of several minutes. Why is that? Well, that's because every cell in your body has to do constant catabolism in order to derive energy, in order to remain viable. This means they need constant food, and particularly oxygen delivery. And if you don't get those things, metabolism can no longer work, and the cells die. And so energy is sort of this mystical concept that we like to talk about. And so before we delve a little bit deeper into what I mean by biological energy, what it is, why we have to constantly do catabolism to maintain it, I want to go back and say just a little bit more about carbohydrates and finish our discussion there, and talk about carbohydrates and sugar polymers, polysaccharides, because this has some additional properties I didn't have time to cover in my prior lecture that is important to understand how it allows these molecules to store energy in a very efficient way. And it will also enable us to talk a little bit about how carbohydrates can also be used as structural molecules for lots of different organisms. All right, so a little diversion to discuss carbohydrates and polysaccharides. And so if you recall from my prior lecture, sugars that are greater than four to five carbons can exist either in open chain or ring forms, furanose or pyranose rings. I'll just remind you by redrawing up here, glucose. So this is D glucose, drawn in the open chain form. Remember this can form a ring. OK. So it's a reminder, carbon 1, 2, 3, 4, 5, 6. If we have this hydroxyl group from carbon 5, form a hemiacetal bond with the aldehyde at carbon 1, you get this ring structure. This would be alpha or beta, depending on whether the hydroxyl group here at carbon 1 is pointing up or pointing down. D-glucopyranose. So this is review of what we already talked about last time. Now, what I want to talk about is that if we form a disaccharide or a polysaccharide-- that is, begin to make sugar bonds, and if we do that in a way that ties up this hemiacetal or hemiketal bond, this then prevents the ring from opening and gaining access to that carbonyl, that aldehyde carbonyl, as this moves between the open chain and the ring form. And so a good example of this is the disaccharide sucrose. So sucrose is a disaccharide of glucose plus galactose. And here is what this disaccharide looks like. OK, so you have here, glucose in the alpha-D-pyranose form. Here's fructose in the beta, because the OH group is pointing out. Fructofuranose form. And so the formal name for this molecule, sucrose, would be alpha-D-glucopyranosyl 1-2, because we're going from carbon 1 of glucose to carbon 2, there's 1, 2, 3, 4, 5, 6 of fructose. Beta-D-fructofuranose. Sucrose or alphaD-glucopyranosyl 1-2 beta-D-fructofuranose.

~09:14OK, so this disaccharide forms a bond between carbon 1 and carbon 2. That would be the aldehyde in glucose, or the ketone tied up in the hemiacetal or the hemiketal bond of these two molecules. And so there's no way that either of these sugars can access the open chain form, without breaking this O-glycosidic bond. And that's one reason why sucrose is a very good storage molecule for carbohydrates, because without breaking that bond, you prevent access to gaining these reactive aldehyde or ketone bonds, that could react with some other molecule in the cell. Now, I mention this because whether or not a particular sugar is enabled to get access to this reactive aldehyde or ketone really forms the basis for a classic sugar detection lab test. And this is relevant to discuss this lab test because it explains some nomenclature that is still used, and in fact, we will continue to use a bit as we discuss some aspects of sugar metabolism. So what this test is, is basically, is that if you heat the sugar with copper, which is blue, and that copper can gain access to a free carbonyl--that is, if the sugar can access the open chain form to expose the aldehyde or a ketone, that copper can then become reduced to copper plus. That changes the color from blue to red. And so if you reduce the copper, you oxidize the bond there. And this test turns positive if there is a reducing sugar, is what it's called. So a sugar that reduces the copper from the blue 2 plus state to the red plus state is a positive test. And so basically, a reducing sugar is any sugar with an ability to access an open chain form. Because that will provide the free aldehyde or ketone to reduce the copper in this test. So this term, reducing sugar, obviously applies to all monosaccharides, because every single one of them has an aldehyde or a ketone, and can access the open chain form. But it will only apply to some disaccharides or polysaccharides. So let's come back to sucrose up there. So is sucrose a reducing sugar? No, it's not, because there is no way that you can access a free aldehyde or a ketone in sucrose, because it's tied up in that O-glycosidic bond between carbon 1 of glucose and carbon 2 of fructose. And so you need to break that bond to give them monosaccharide subunits in order to access the open chain form. However, let's give an example of a disaccharide that is a reducing sugar. And that's the disaccharide maltose. So maltose is basically two glucose molecules. And those two glucose molecules are like this. So this has a O-glycosidic bond between carbon 1 of this glucose, and carbon 4 of that glucose. And so formally, this would be alpha-D-glucopyranosyl 1-4 alpha or beta. If it's pointing down, it's alpha, pointing up, it's beta. Dglucopyranose. So maltose or alpha-D-glucopyranosyl 1-4 alpha or beta D-glucopyranose is a reducing sugar, because this sugar could access the open chain form. You could open up this carbonyl and expose the free aldehyde at position 1. Now, we could also refer to this sugar as having two different ends. We can refer to this end as being the nonreducing end, and this end as being the reducing end. Because of course, this bond, this aldehyde on the first glucose is tied up in this O-glycosidic bond, whereas the one on this one is not. So this end is non-reducing, and this end is reducing. Now this becomes much more relevant if we start talking about polymers. And so what starch is. So starch, what potatoes are made out of, is basically a polymer of maltose. Polymer of maltose molecules. So basically our glucose with 1-4 linkages.

~12:39OK, so I could draw that end, the reducing end of the polymer, in the alpha or beta. But this here, being the nonreducing end. Every other subunit is tied up in this 1-4 O-glycosidic bond. And so starch is really a polymer of glucose molecules with bonds between the 1 and the 4 position of each subunit. Now we will see later that we build and break down starch polymers from only one end, from the non-reducing end. And so having these terms, reducing and non-reducing, provides the term to specify different ends of a polymer. Reducing and non-reducing ends turns out to also be important for naming conventions for disaccharides or polysaccharides. And this becomes relevant if we consider the disaccharide lactose. So lactose is also a polymer of glucose plus galactose, except this polymer of glucose plus galactose, unlike sucrose, is different because it has a different linkage between the sucrose is a disaccharide of glucose plus fructose. Fructose, I wrote the name correctly. But lactose is a disaccharide between glucose and galactose. And so I'll draw it here. OK, so this is lactose. This is galactose here on the non-reducing end. So the formal name for this would be beta, because there's a beta linkage here between carbon 1 of this galactose and carbon 4 of this glucose. So be beta-D-galactopyranosyl 1-4. And this is glucose in the alpha or beta. D-glucopyranose. Lactose, or beta-Dgalactopyranosyl 1-4, alpha or beta, depending if I drew down, alpha, up, beta. D-glucopyranose. Now, this also has non-reducing and reducing ends. And by convention, you would name the sugar from the nonreducing end to the reducing end. Hence I put galactopyranosyl 1-4 alpha-D-glucopyranose, named them in that order. Now you'll note that lactose, unlike starch, unlike maltose, unlike sucrose, links these disaccharides with a beta linkage between this sugar and that sugar, where the other ones had alpha linkages. And structurally, this is very different. And I just want to point that out. So this here is basically alpha-D-glucopyranose. And so alpha, this is carbon 1. This is the hydroxyl pointing down. That's why it's alpha. If I make an O-glycosidic bond to another sugar, you can see that it points and creates this kinked structure. Now, if I were to make this beta, this hydroxyl rather than being here would be at this position on carbon 1. You can see that that now creates a very different geometry. Now you have a flat molecule, as opposed to a kinked molecule. Now, this has consequences, of course, for the enzymes that break down the sugars. Obviously, it's going to be a very different enzyme that breaks a bond in this orientation, versus a bond in that orientation. This has, basically implications for what enzymes we have. And so lactose, of course, is milk sugar. All mammals make milk, so make lactose, and break it down when we're babies. But most mammals tend to lose expression of the enzyme lactase that's able to break this 1-4 beta linkage as we age. And this is basically what accounts for lactose intolerance. Now, if you think about it, in the world, much of the world has issues with varying degrees of lactose intolerance in adulthood. The exception to this is often people of European ancestry. Why is this?

~15:36Well, Europeans historically drank milk well into adulthood. And so this selected for continuous lactase, the enzyme that wouldn't break that beta 1-4 linkage. The expression as people move into adulthood. And so it's an example of how cultural things, drinking milk into adulthood, really affected evolution, such that that subpopulation of people, people with that genetics to increase lactase expression, don't become lactose intolerant as they age. Now, this alpha versus beta linkage-- that is, whether or not you have this more kinked versus more flat structure-- also has a major effect on the structure of a polymer as well. And so if we look here at this starch polymer, it's basically this series of glucose molecules stuck together with alpha 1-4 linkages. So that's going to create a kinked structure. This is best shown here on this slide. So you can see this kinked structure. And so when you build this polymer, you're going to end up more with this helical structure in 3D space. That's a very efficient way to store glucose monomers in much less space than you would otherwise get with beta linkages. Now, it turns out that nature takes this a step further. And in addition to just having this starch polymer, it turns out sometimes you can add branches to the starch polymer to further increase the efficiency of energy storage. So if you have here a starch polymer, so let me just draw here a couple subunits. So this here is a starch polymer, glucose polymer with alpha 1-4 linkages that I can put an additional branch on this by adding on a linkage up here on the top. With an alpha 1-6 linkage. So the 1 position of here, to the 6 position of here, which has the effect of creating branches on this long polymer, such that you have a single reducing end, and lots of non-reducing ends. Each of these non-reducing ends has a polymer of alpha 1-4 linkages that would be stuck to the main chain polymer via this alpha 1-6 linkage. And this has a couple implications for storage. The first is, by only having one reducing end, it reduces maximally the exposure to a free aldehyde. And so that makes it a good storage sugar. And also as we will see, we're going to break down these sugar polymers biologically from the non-reducing ends, and build from those ends. And so there's lots of hooks or lots of places to either add or remove sugars from, which allows you to access carbohydrates quickly as needed. That's shown nicely here on this slide, better than I can draw it. So up here, you have your non-reducing end across the polymer. So this here would be starch with the alpha 1-4 linkages on the polymer, or reducing end on one side, non-reducing end on the other. You can then add branch points to that by making an O-glycosidic bond between carbon 1 down to carbon 6, creating a branch. And so you can see this helical branch structure will then form. That is both very efficient to pack lots of glucose into one place, and give lots of non-reducing ends with which to build and remove sugars from for biology to either store or quickly access sugar molecules as needed. Now, in plants, of course, potatoes use starch in a straight chain. We know that. However, plants also use this branched structure. This is a molecule called amylopectin. Amylopectin is better known in the food industry as Sure-Jell. It's the material that allows you to make jelly. It's the jelling agent in jelly. And effectively, this has every 24 to 30 units. There would be a branch with one of these alpha 1-6 linkages to give you this multi-branched structure.

~18:57Now, animals don't make starch. Animals don't make amylopectin. But animals make another molecule called glycogen. Glycogen, also a sugar polymer, exactly like amylopectin. Alpha 1-4 linkages with the alpha 1-6 to create branch points. Just like amylopectin, except glycogen has even more branches with a branch every 8 to 12 glucose units. And so, this is a very complex structure for both plants and animals to quickly store and quickly release glucose molecules when they're needed. Now, this is in contrast to a glucose polymer that can be done that has a very different 3D structure. So what if we take starch, and rather than have these alpha 1-4 linkages, but instead replace these with beta 1-4 linkages? OK, so now beta linkages. Looks simple enough, but changing the geometry from the alpha to the beta bond removes the kink. Now it's much more of a flat structure. Turns out this is, shown on an image here. So here's a beta linkage, and suddenly you have this flat structure. This is the same polymer as starch chemically, but has a different linkage, the beta 1-4 linkage. And that polymer with the beta 1-4 linkage is cellulose. And cellulose is, of course, what wood is made out of. And so wood and potatoes, cellulose and starch, exactly the same polymer. Same number of calories, if you could access the glucose units. But the alpha and beta bonds make them very different. Obviously, wood and cellulose is a very good structural polymer for plants. We build houses out of it. We don't build houses out of potatoes, but we eat a lot of potatoes. So equally good molecule for plants to store lots of food for energy. Turns out wood is a great source of energy too. You just need to have an enzyme that can break the beta 1-4 bond. Termites have symbiotic microbes that allow them to do this, and this is why termites can eat wood, a lot of energy tied up in wood. Now, we lack the time to go into the details about how this relates to other structural carbohydrates. But in general, structural carbohydrate or carbohydrate-like molecules form polymers of sugar or related molecules, also with these beta linkages. And a good example is chitin, so the material in the insect shells, is basically a sugar-like molecule that's a polymer with a beta linkage in it. Same thing with cartilage in humans and animals. And you can look up the details of what these things look like, of course, if you're interested. Chitin and cartilage aren't made of true polysaccharide polymers, but they're very related to polysaccharides. And they really illustrate how nature can take carbohydrate chemistry and repurpose it to basically act as an energy source, but also build all kinds of structural molecules that are really useful in biology. All right, now I want to go back to, here's ways that one can store and use carbohydrates in interesting ways. But now I want to come back to them as energy sources, and assuming you can access the glucose and whatever polymer it's present in, how can you actually metabolize it to provide energy in a way that sustains life? And this means we're going to turn to another topic, a topic that's typically referred to as bioenergetics. Bioenergetics, which is really the discussion of how energy is transduced in biological systems.

~21:58I'm going to introduce this topic today, we're going to revisit it throughout the course. But it's really important to ask this question. We really want to consider, what do we mean when we say biological energy? What is biological energy? Well, someone probably made you memorize in high school, and often you were thinking, oh, it's just ATP. And certainly, ATP, adenosine triphosphate, is a very important molecule for biological energy transduction. But I want to actually explore why ATP is actually useful. And if ATP is energy, why don't we just eat all kinds of ATP? I can say for a fact that absolutely none of you had ATP today for breakfast. No one sells ATP as an energy booster. And so if ATP is such a great energy source, why don't we just eat it? And to really understand ATP, how it works, what biological energy really is, we really need to revisit some very basic topics in thermodynamics.

~22:50Now, thermodynamics strikes fear into the hearts of students everywhere. You can get details of thermodynamics and the theory around these things in other courses. That's not really what we're going to try to accomplish here in 705. Our goal here is really a practical understanding of how thermodynamics applies to biology and metabolism. And we need to go into this, because it's key to understanding why we store energy, go through all this trouble to store energy as these carbohydrate polymers to begin with. Why we eat potatoes and not ATP, as well as why ATP is actually useful to cells. And so let's take a step back and just think about it. Let's think about wood. I just told you, wood is a polymer of glucose molecules with beta 1-4 linkages. How can we, forget as organisms, as cells. But how can you release energy from wood, to do other kinds of work? Well, we can burn it. And so what's the chemistry of burning wood? Well, it's a carbohydrate polymer. So it has this CnH2n formula. If we combine that carbohydrate with oxygen, it releases CO2 and water, plus some light, plus heat. This light and heat is energy, because we can use it to do work. Boil water, turn a turbine, make electricity. Whatever. Warm ourselves by the fire. Use the light to do some other things. And so this burning of wood is certainly release of energy. Now, we lack the enzymes to do this reaction in wood, because we can't break that beta 1-4 linkage. But we certainly can break that alpha 1-4 linkage in starch from potatoes. And we use the exact same chemistry to burn that glucose and release energy. Except the difference is that if I burn wood, I do this all in one step. But life is a much better engineer than that. It basically, rather than releasing all of that energy in one step, it really releases it stepwise in a manner that's actually useful to cells. But it's exactly the same release of energy. And so what life is, or extracting biological energy is, is it's really the ability to do stepwise oxidation of glucose or other carbon. I'll write glucose for now. To get energy. So we burn wood, that's favorable, it releases energy. If we do stepwise oxidation, we also can release energy as well. Same reaction, also releases energy.

~24:59How much energy do we get from releasing us burning glucose, as you do and burning it in a fire? Well, it has to be exactly the same amount, because it's exactly the same reaction. So we burn wood, we get light and heat. Cells burn glucose, it releases the exact same amount of energy. It just does so in a way that allows the cells to do biologically useful things. What are those? Well, it could be heat. All of us maintain temperature. So heat is a conversion of energy that of course, is useful to biology. But it can do other things, too. It can allow cells to move. It can allow them to do any reaction to fight that entropy that allows life to exist. And so all things, including ourselves, have to follow the same laws of thermodynamics. Life is not special in that regard. And so burning wood is favorable, because it increases the entropy of the universe. You're breaking up this polymer into a bunch of monomers. That's going to be spontaneous. That is in accordance with the second law of thermodynamics. True if cells burn it as well. But remember, the second law says that net entropy of the universe must always increase. And so if we're going to do something that's not energetically favorable, like build a glucose polymer, we obviously have to put energy into it. And to do so, the energy that's released must be greater than the energy that we actually store. And so this is actually a really key point, because anything that we do that requires energy input for a cell to carry out that process requires a source of energy release somewhere else that is equivalent or greater than what is actually put in. And so cells need energy, because they do lots of thermodynamically unfavorable things, fighting that entropy of the universe. And all of those processes must come from the release of energy elsewhere, like the burning of carbohydrate. And so that constant energy input is at the very highest level, why we need to do constant metabolism in order to maintain order and survive as organisms. Now this, of course, comes from things like burning of carbohydrate. But of course, that carbohydrate has to come from somewhere. And so ultimately, you have to have an external source. That external source is of course, the sun. Photosynthetic organisms can use light energy from the sun to do exactly the same things. Build those excess polymers, and that's why we as animals or anything that eats other animals as a way to live is ultimately depending on eating photosynthetic organisms, because photosynthetic organisms do this by harvesting energy of the sun. Now that's all very high level. But I want to come back to the specifics of metabolism, ultimately, and understand those as how they relate to enzymes' reactions and pathways. That is, how do you get that complex series of reactions that make life possible, and ultimately still be guided by these exact same principles? In other words, how do we couple the energy releasing reactions, like burning glucose, in a way that obeys the laws of thermodynamics and operates under biologically acceptable conditions in order to do things that are biologically unfavorable. That's really what bioenergetics is. And so the next thing we want to consider then, if we want to understand this, is really, let's ask the question. What determines if a specific reaction occurs? That is, what determines if something like burning wood actually spontaneously happens?

~28:08If you light wood on fire, it will burn. It does so every single time, everyone has had that experience. But no one has ever seen CO2 and water spontaneously come together and form a tree. That doesn't happen. So what determines whether you burn wood and that is favorable, versus why it is that CO2 and water don't come together and spontaneously form a tree? Do not think that this is determined entirely by enzymes. Enzymes are catalysts. Enzymes are the spark that makes the burning of wood possible. But remember, enzymes cannot change thermodynamics. Enzymes only change the rate at which a reaction happens. It does not change the equilibrium. That tree wants to, if given the catalyst, form CO2 and water. That reaction is spontaneous because it is thermodynamically favorable. That tree will not take CO2 and water and spontaneously reform a tree. And there's no amount of enzyme that will make that happen all by itself. And so remember that. If you have any reaction between A and B, there is some equilibrium, as defined by the chemical properties of A and B, such that the equilibrium lies far to one side or the other. And that equilibrium is determined by thermodynamics. It is not determined by enzymes. I don't care how much enzyme I add. If the equilibrium lies towards A as I draw it, I can add all the enzyme I want, and I can never create more B. And so, this is super important, and is actually a point that is gotten wrong by lots of biologists. They think, oh, this enzyme is expressed. Therefore, this pathway must be happening, or this reaction must be happening faster. You need an enzyme to catalyze a reaction. So a reaction may not occur, your wood may not burn unless you give it a spark, a catalyst to actually make it happen. But you cannot add a catalyst to fight thermodynamics. You cannot take that CO2 and water and turn it back into a tree. And the same thing is true. You can add all the enzyme in the world you want, and all it will do is help A and B establish the equilibrium that is defined by the thermodynamics of the relationship between A and B. Now, what determines the equilibrium between any two species? That's a topic for another class, why a species is favored to be one side or another. However, a useful tool to think about this in biochemistry and quantify this for any pair of reactions is something you've probably learned about before. It's something called the Gibbs free energy. Gibbs free energy, or delta G. Delta G is related to the way we'll talk about the equilibrium constant. And it's basically a term that we can use to specify if a reaction is favorable or not. And so hopefully you learned in an introductory class that if delta G is less than 0, our reaction is spontaneous. If delta G equals 0, our reaction is that equilibrium. If delta G is greater than 0, our reaction is not spontaneous. All right, if we take our log and ask, what is delta G to turn that log into CO2 plus water, it is less than 0, because it is very spontaneous. We take a pile of ashes and ask what delta G is to recreate the log. It's greater than 0, because it ain't going to happen. And if it's at some equilibrium, delta G is equal to 0. Now, delta G depends entirely on conditions, as we will see in a minute. And because of that, this means that there can be conditions where absolutely any reaction can be favorable if the conditions are right. And we'll see that this becomes very relevant to understand how metabolism works. And so let's go back and just now consider some generic reaction, A and B.

~31:37So at equilibrium, what happens? So at equilibrium, the concentration of A and the concentration of B are not changing. Delta G equals 0. These two things are at equilibrium, whatever that equilibrium is. A and B are not changing in concentration. Now if I come and I add more A to this side of the equation, what does that mean? Well, you know from Le Chatelier's principle that that's going to favor production of B. We have shifted things now out of equilibrium. So if I add A, that leads the production of B to re-establish this equilibrium. That means that delta G from A to B is less than 0 until I've reestablished equilibrium, and now delta G is back to 0 again. Now, what happens if I add B? Well, if I add B, now I'm going to produce some A. So add B. That leads the production of A, until I reestablish that equilibrium. So delta G, in that case, to go from B back to A. The reverse reaction is less than 0. Or I could also say that delta G, to go to A to B here, because that's not going to happen. I just added more B. I'm not going to suddenly create more B from A. So here now, delta G is greater than 0. Now note when I did this, I don't actually have to specify how much A or how much B I've added. The equilibrium depends on the ratio of B to A. Not the absolute concentration of either species. And so having a way to think about this equilibrium, it turns out, is also useful for biological systems to divine some delta G that is helpful to relate to this equilibrium constant. And that is this concept of delta G 0 prime. A standard free energy, which for biological systems, relates to the equilibrium constant at 25 degrees, or the pH of 7 and 1 atmosphere. Pretty typical biological conditions. And this here is basically related to the equilibrium constant. And we can calculate the actual delta G by the following formula. Delta G equals delta G 0 prime plus RT times the log of the products over the reactant. So this would be drawn for the reaction A to B. So the reaction A to B is related to delta G 0 prime, which is related to the equilibrium constant. I'll tell you how in the second. Plus R is the gas constant, T is the temperature in Kelvin times the natural log of the ratio of products, B over reactants, A. This tells you whether or not at specific concentrations of B and A, that is a specific ratio of B and A, whether it is favorable to go from A to B, delta G less than 0, or whether it is favorable to go from B to A, delta G greater than 0. And so this should begin to make it clear that whether or not a specific reaction occurs is affected by the actual conditions present. And you can calculate that based on this relationship. And so if I specify concentrations of A and B, as well as delta G 0 prime, I can know, at those concentrations, which direction of the reaction is favored. Now of course, my drawing A to B is entirely arbitrary. I could equally draw it B to A. And if I did that, so this is delta G 0 prime, A to B, all I do is flip the signs. So delta G equals delta G 0 prime. The negative of A to B is equal to the positive of delta G 0 prime from B to A, because the direction is arbitrary. Plus RT log in this case. Now I have A is my product, and B is my reactant. If I just flip the sign, flip that ratio, it's just going to change the sign of my product and give me exactly the same result with the opposite sign, which makes sense. What's favorable in one direction is not favorable in the other direction, and vice versa.

~35:17Now, I can also set delta G equals 0. That's equilibrium. If I do that, then delta G 0 prime equals negative RT. So this would be for A to B. I'll use the top example. So delta G equals 0. Then I have delta G 0 prime equals RT times log B over A. So if I know two things are at equilibrium, I can calculate delta G 0 prime, and know what the equilibrium constant is. And so it follows from this then that if delta G 0 prime is negative, that means B is favored over A at equilibrium. And if delta G 0 prime is positive, that means A is favored over B at equilibrium.

~35:55And so delta G 0 prime really is a convenient way to look at a reaction, and know which direction the equilibrium lies. So A and B, delta G 0 prime is negative, equilibrium lies to B. Between A and B, if delta G 0 prime is positive, the equilibrium lies towards A. But the key concept is that for any specific reaction, any specific conditions of A and B, whether or not A is converted to B will be defined by the equilibrium constant. But it will also be defined about what delta is under those conditions. So to be clear, delta G will depend on conditions, because I'll write it again, delta G equals delta G 0 prime plus RT times the log for the reaction, A goes to B. So products over reactants. And so whether or not A is converted to B will be a property of the equilibrium constant, plus the concentration of B and the concentration of A, that ratio of concentrations at the conditions present. And if in that calculation, delta G is less than 0, that reaction is spontaneous. If delta G is greater than 0, that reaction is not spontaneous. Or to put it another way, if delta G is less than 0 under those conditions, energy is released. Or if delta G is greater than 0, that reaction will not occur without energy input. If I burn wood, energy is released. But there's no way I can reassemble that log without some kind of energy input. And really getting this concept is central to understanding metabolism, as well as what biological energy means. And hopefully, what is now apparent, based on what I just said, is that what really determines delta G is two things. Of course it's the equilibrium constant. But in essence, delta G for any reaction is proportional to the ratio of the reactants over the products. And that I can come up, at least in theory, with any relationship, any ratio of reactants to products to make this favorable. At some tiny, tiny concentration, if I have nothing but CO2 and water, and infinite time and infinite catalysis, a tiny bit of wood could spontaneously form. And so life really creates the conditions that allow that, and select for that to happen, and of course, add energy input so it can actually happen more. And so this should be very clear to you that absolutely no reaction is irreversible. I don't care what your textbook says. Lots of sources will discuss irreversible reactions. I'll talk about irreversible reactions later in the course. But what we mean when we say the word irreversible reaction is these are under conditions that are found in cells and in nature. And I stress this because if we want to understand the energetics for how pathways work, we have to appreciate that life can create conditions to do unfavorable things, because that's the only way that pathways can work.

~38:33All right, what what do I mean by that? Well, suppose again, we'll come back to our reaction. A goes to B. Suppose I need to build a pathway where I need to convert A to B, because B is actually useful for some purpose that I need to do in a cell. However, what if this is at least by equilibrium, unfavorable? What if the equilibrium lies towards A? That means delta G 0 prime is positive, is greater than 0, for A to B. What does that mean? Delta G 0 prime greater than 0, for the reaction A to B? That means the equilibrium lies towards A. And so there is no amount of enzyme I can add that will allow me to net convert A to B, because the equilibrium lies far towards A. However, whether or not a cell is able to build a pathway, where it converts A to B depends on delta G, not the equilibrium constant for that specific reaction. So how can I turn A into B? Well, I could turn A into B if I keep the concentration of B low enough that delta G, not delta G 0 prime, that's the equilibrium constant, but delta G favors A to B conversion? That is, delta G for A to B is less than 0. How can I do that? Well, remember delta G equals delta G 0 prime plus RT log of B over A. So this term is positive, because I told you, the equilibrium lies towards A. So I need a concentration, a ratio of B to A, such that B is low enough that this term is more negative than that term is positive. And if that's the case, delta G will be less than 0, and I can net move that forward. So how can I do that? How can I keep the concentration of B low? Well, I can build a pathway that consumes B in a way to keep it low, which favors A to B conversion. In other words, I can build a pathway, A goes to B goes to C. So even here, where delta G 0 prime is greater than 0, if delta G 0 prime for this reaction, B to A, is much less than 0, such that equilibriums strongly favor C, that means my unfavorable equilibrium here can be overcome by the very favorable equilibrium of B to C. In essence, I can use B to C to pull A to B, such that it actually happens. Produce this intermediate that's useful on the path to making C. This strategy is used in lots of metabolic pathways. And it's useful to generate lots of chemically useful intermediates. Now note that this only will work if conversion of A to C is favorable. That is, if delta G 0 prime is less than 0. I can only pull this reaction if delta G 0 prime is less than 0. That is, if equilibrium favors A to C, I can build a pathway this way to do something unfavorable on the way to making C. This brings up another key point, in that everything that we discuss, relationship between any two metabolites in any single reaction, also must be true for entire pathways. So I have a three-step pathway from A to C. Whether I turn A into C in one step, or turn A into C in multiple steps, the free energy is exactly the same. If I burn wood by lighting it on fire, it releases the same amount of energy than if a termite has an enzyme that breaks that beta 1-4 linkage in the wood, and can burn that glucose stepwise through metabolism, the exact same amount of energy is released. Glucose on one side, CO2 and water on the other side, exactly the same amount of energy released, whether I do it in one step, or I do it in multiple steps. The equilibrium constant between those has to be the same, and the delta G has to be the same between all of it.

~42:13And so it should become apparent, then, that A to C has to be favorable if I'm going to use this trick of keeping product low in order to pull an otherwise unfavorable reaction forward. In other words, there's no way for me to keep B low and net convert A to B all on its own. That will not work. And it's these unfavorable reactions that ultimately then require energy input. And this is where ATP now starts becoming useful for cells. OK, so now I want to talk a little bit about how ATP provides energy that allows otherwise unfavorable reactions to occur. And ATP is useful because delta G applies to sets of reactions in the same way it applies to single reactions. That is, it applies to whole systems. All reactions are a series of reactions, whether they happen alone or coupled together. All have to follow the same rules. So now let's make our reaction more complicated. Let's say A plus B goes to C plus D, where this is really two reactions coupled together. A being turned into C, and B being turned into D. All right, so will this reaction happen? Can I turn A plus B into C plus D? Well, how do I know? Well, it's going to be, got to calculate delta G. And so it's going to be delta G 0 prime for A going to C, plus delta G 0 prime for B going to D. So those will relate to what's the equilibrium constant, the equilibrium relationship between A and C and B and D. If it's less than 0, equilibrium will favor to the right. If it's greater than 0, equilibrium will favor to the left of A and C and B and D. Add them together, that gives me my overall delta G 0 prime for those two reactions. Plus RT times the log of the products. So that's C and D over A and B. Products over the reactants. So if I take an unfavorable reaction-- say, A to C, if delta G 0 prime is greater than 0, so it would favor A, and I couple it to another reaction, B to D that's very favorable, because I add these two terms together. I can now take something where the equilibrium would be unfavorable, and make it favorable. However, whether or not this actually happens still depends on this ratio of products to reactants. So it still depends on the ratio of C to A, and D to B in our hypothetical thing here. So to know if a specific reaction will actually occur, we have to take into account both the sum of the delta G primes related to the equilibrium constant, and the actual ratios that are present in cells. And so the net effect is that I can couple more favorable reactions to make otherwise unfavorable reactions now become favorable. That is, have energy input. But it still depends on conditions. And so let's think a little bit about the polymer synthesis that we learned from Professor Yaffe. And so in that process, what did we do? We made several polymers, and you learned about the chemistry to do this. We made DNA, we made RNA, and we made protein. These are polymers of nucleic acids. Protein is a polymer of amino acids. All of these fight entropy, right? We're building polymers, not breaking them down. And all of them were synthesized using reactions that hydrolyzed ATP. In fact, those reactions actually hydrolyzed ATP in the following way. And every single one of those cases, if you look back, what you will see is that they all had steps where ATP was taken to AMP plus 2 inorganic phosphates. This reaction is very favorable. In fact, it's two reactions. It's really ATP goes to AMP plus pyrophosphate.

~45:39And then that pyrophosphate goes to 2 inorganic phosphates. It turns out that delta G 0 prime is less than 0 for both of these reactions. That means the equilibrium lies towards AMP plus pyrophosphate. And here, the equilibrium lies towards 2 inorganic phosphates. And so there's actually two tricks here by metabolism. One is, it's two very favorable reactions that we're coupled to do something unfavorable, build a polymer. And use the trick of keeping pyrophosphate low by coupling it to a downstream, very favorable reaction, which even further pulls this reaction, and is why ATP hydrolysis here becomes so useful as a way to build these polymers. Let's just go quickly and add a few numbers to show exactly what I mean by how ATP hydrolysis can be useful in this setting. And so, let's do something a little simpler. Let's go to ATP plus ADP plus to inorganic phosphate. So let's give this some numbers, delta G 0 prime in this case is equal to negative 7.5 kcals per mole. All right, what does this mean? Delta G 0 prime is related to the equilibrium constant. It's less than 0, which means the equilibrium lies towards ADP plus phosphate. Now let's see how this actually helps by considering what the first step in glucose metabolism by cells is. So the first step cells do in glucose metabolism is add an inorganic phosphate group to glucose. So it's this reaction, glucose plus inorganic phosphate goes to glucose phosphate. Just a quick note on shorthand that I will use in this course. So PI, of course, is inorganic phosphate. That is this PO43 minus group. When it forms a phosphodiester bond, like to an alcohol and one of the glucose molecules, I'll draw this in a future lecture. I'll oftentimes indicate it by putting a circle around the phosphate. And so adding this phosphate to glucose is a step that traps that glucose in cells. For now, let's just consider this reaction. And so delta G 0 prime of this reaction is positive 3.3 kcals per mole. What does that mean? Delta G 0 prime relates to the equilibrium constant. It says the equilibrium constant lies here to the left, to the glucose plus phosphate side. So if we want to net trap glucose phosphate in cells, well, it's not going to happen spontaneously. You need some kind of energy input. That can come from ATP. And so if we couple those two reactions as follows. So we now have glucose plus ATP goes to glucose phosphate plus ADP. Now let's draw this out. Will this happen? So we can calculate delta G. Delta G equals delta G 0 prime. So there's two reactions happening here, glucose to glucose phosphate, and ATP to ADP. We know what delta G 0 prime is by adding those together. So it's 3.3 plus negative 7.5. That equals negative 4.2. What does that tell us? That says, now we've created conditions where the equilibrium constant lies towards the right, towards the glucose phosphate plus ADP side. But how much of this occurs or whether it occurs isn't just the property the equilibrium constant. It's also how much is there. And so that's RT times the log of the products, glucose phosphate.

~48:34And ADP over glucose. And ATP. And so, if this term is less than 4.2-- that is, if the ratio of glucose phosphate to glucose and ADP to ATP in this term ends up being less than positive 4.2, this reaction is spontaneous. It is favored. Lots of conditions where that would be the case, although if this term is 4.2 or greater, now it will no longer occur. How much ATP is needed? Well, it's hard to answer this question in absolute terms, because if there's a lot of ADP around, you need a lot more ATP for the ADP to be useful. And this is the problem with equating ATP with energy. The energy here is in the ratio of ATP to ADP, or ADP to ATP. And this is something that we we'll come back to in the next lecture, because in the end, this is what will determine if a reaction has happened. It is not the absolute concentration of ATP. It's the fact that ATP hydrolysis is favorable. But how favorable that is depends on the relevant concentrations of ATP or ADP. And relative concentrations are all that matters in thermodynamics. It is not absolute concentrations. It is the ATP-ADP ratio that is the correct way to think about energy. And this is true for any reaction with respect to energetics. If an ATP-ADP ratio exists, such that when coupled to a reaction, delta G is less than 0, then it becomes spontaneous. And this is how ATP provides energy. And we will come back to this more in the next lecture. Thank you.

1.3同多糖的构象分道扬镳与多糖动员策略：土豆与木头、加磷酸解与水解的进化博弈

一、多糖的通性与同多糖的几何分水岭：为什么 α-1,4 盘成螺旋，而 β-1,4 伸成直链？

当成百上千个单糖通过糖苷键聚合在一起，就形成了多糖（Polysaccharides）。在进入具体多糖之前，请先在脑海中刻下多糖与蛋白质、核酸截然不同的三大通性：

1. 无模板合成与分子量多分散性：蛋白质合成有 mRNA 作模板，核酸复制有母链作模板，因此纯化的每条多肽或 DNA 都有精确均一的相对分子质量；而 多糖的生物合成没有模板指导，全靠糖基转移酶催化延伸，因此即便是纯品多糖也没有确定的相对分子质量，分子大小不均一（呈多分散性）。
2. 宏观无还原性与无变旋现象：一条由上万个葡萄糖残基组成的多糖链，虽然在链的最右端理论上仍有 1 个游离异头碳（还原端），但 1/10000 的比例在化学检测中完全可以忽略不计！因此，多糖未经水解时不表现还原性，无变旋现象，无甜味，一般不能结晶，大多不溶于水或仅形成胶体溶液。
3. 命题大坑：多糖有旋光性吗？ 千万注意：多糖虽然没有变旋现象（因为半缩醛羟基几乎全被锁死），但多糖分子内部含有成千上万个手性碳原子（C^*），因此多糖具有显著的旋光性（例如淀粉和右旋糖酐都具有很强的右旋光性）！记住学长口诀：“变旋看半缩醛羟基，旋光看手性碳”！

由同一种单糖（或单糖衍生物）聚合而成的多糖称为同多糖（Homopolysaccharides）。在 L19 课堂上，Vander Heiden 教授在投影仪上并排展示了淀粉（Starch）和纤维素（Cellulose）的分子模型：两者 100% 都是由 D-吡喃葡萄糖通过 (1→ 4) 糖苷键串联而成，唯一的区别仅仅在于 C1 异头碳的构型一个是 α(1→ 4)，一个是 β(1→ 4)！为什么这小小的一键之差，会让土豆变成软糯的口粮，而让木材变成造房子的钢筋铁骨？

3D3D 糖类：同多糖（淀粉、糖原、纤维素与壳多糖）交互观察直链淀粉 α(1→4) 左手螺旋包埋碘显蓝色、支链淀粉与糖原 α(1→6) 分支及 n+1 个非还原端、纤维素 β(1→4) 氢键微纤维与几丁质↗

3D3D 糖类：同多糖（淀粉、糖原、纤维素与壳多糖）交互观察直链淀粉 α(1→4) 左手螺旋包埋碘显蓝色、支链淀粉与糖原 α(1→6) 分支及 n+1 个非还原端、纤维素 β(1→4) 氢键微纤维与几丁质↗

二、五大同多糖全景拆解与“为什么糖原每 8–12 个残基就要分叉？”

让我们结合王镜岩教材与 MIT 7.05 课堂推演，逐一梳理五大核心同多糖的考点细节：

1. 淀粉（Starch）——植物的能量储库：
   天然淀粉颗粒由约 20%～ 25% 的直链淀粉（Amylose） 和 75%～ 80% 的支链淀粉（Amylopectin） 组成（二者之比约 1:4）。

• 直链淀粉：纯 α(1→ 4) 不分支长链，仅少量溶于热水，遇碘显深蓝色。
• 支链淀粉：主链为 α(1→ 4) 糖苷键，分支点为 α(1→ 6) 糖苷键，平均每 24～30 个葡萄糖残基出现一个分支 2025·填空，相对分子质量可达 200 万以上，易溶于水形成稳定的胶体溶液（在食品工业中用作果冻胶凝剂，也是直链淀粉的保护胶体），遇碘显紫～红紫色。

1. 糖原（Glycogen）——“动物淀粉”与极限分支的工程学奇迹：
   人体糖原主要贮存于肝脏（占肝湿重可达 7%～ 10%，用于维持餐间血糖恒定）和骨骼肌（占肌湿重 1%～ 2%，但因全身肌肉总质量大，肌糖原总量反而更多，专供肌肉自身收缩耗能）。在细胞质中，糖原与合成酶、分解酶及调控蛋白共同组装成直径 10～ 40 nm 的 β-糖原颗粒。

• 结构特征：糖原与支链淀粉结构高度相似，均以 α(1→ 4) 糖苷键形成主链、以 α(1→ 6) 糖苷键形成分支点，但糖原的分支比支链淀粉更密集、支链更短——平均每 8～12 个葡萄糖残基就有一个 α(1→ 6) 分支 2024/2025·填空！
• Vander Heiden 教授课堂两大设问：
• 设问 A：为什么细胞不直接把葡萄糖以游离单糖形式存在细胞质里，非要费劲耗能合成糖原大分子？
  答（渗透压生死战）：肝细胞内贮存的糖原若折算成游离葡萄糖，其浓度高达 0.4 mol/L（400 mmol/L）！而细胞正常生理渗透压仅相当于约 5 mmol/L 血糖；渗透压只取决于溶质颗粒的摩尔浓度而与分子大小无关！将数万个葡萄糖聚合为 1 个不溶于水的高分子糖原颗粒（浓度仅 0.01 μmol/L），使胞内渗透压暴降数万倍；若以等量游离葡萄糖贮存，外界水分会瞬间涌入把肝细胞活活胀破（渗透裂解），同时 400 mmol/L 胞内葡萄糖也会造成极大的逆浓度梯度，使细胞根本无法再从血液（5 mmol/L）中摄取葡萄糖！
• 设问 B：既然直链淀粉也能降低渗透压，为什么动物的糖原要进化出每 8～12 个残基就分叉一次的超密集树冠结构？
  答（创造海量非还原端，实现秒级并发动员）：仔细数一数分支拓扑学——在一个糖原大分子中，不管分叉多少次，永远只有唯一 1 个还原端（深埋在核心的生糖原蛋白 Glycogenin 上），但每增加 1 个 α(1→ 6) 分支，就会凭空多出 1 个游离的非还原端（即若有 n 个分支，就有 n+1 个非还原端）！
  在生物体内，负责拆解糖原的糖原磷酸化酶（Glycogen phosphorylase）和负责合成糖原的糖原合酶（Glycogen synthase），都只能识别并从非还原端（Non-reducing ends）开始逐个加减葡萄糖残基！如果糖原是一根不分支的长链，10000 个葡萄糖只有 1 个非还原端，每次只能让 1 个酶分子慢吞吞地排队啃；而每 8～12 个残基分叉一次，就能在颗粒表面暴露出成百上千个非还原端，让成百上千个糖原磷酸化酶分子同时扑上去并发切糖，在遇到天敌逃命或剧烈运动的一瞬间爆发出惊人的供糖速率 2024·填空！此外，仅留 1 个被掩埋的还原端也彻底消除了游离醛基的化学毒性。

1. 纤维素（Cellulose）——生物圈最丰富的有机碳库：
   占植物界碳总量 50% 以上，是地球上最丰富的有机物质。由 10000～ 15000 个 β-D-吡喃葡萄糖通过 β(1→ 4) 糖苷键连接而成的不分支线型同多糖 2008/2011/2012·填空 2009/2015·单选。人类和大多数高等动物消化道只分泌水解 α(1→ 4) 键的 α-淀粉酶，完全缺乏水解 β(1→ 4) 键的纤维素酶（Cellulase），因此不能消化纤维素获取能量（但作为不溶性膳食纤维可刺激肠道蠕动）；唯独白蚁肠道内的共生原生动物（披发虫）和反刍动物（牛、羊）瘤胃中的共生细菌能分泌纤维素酶，帮宿主把木头和青草变成葡萄糖。
2. 壳多糖（Chitin，又称几丁质）——自然界第二丰富的多糖：
   广泛构成昆虫、虾蟹等节肢动物坚硬的外骨骼以及绝大多数真菌的细胞壁。壳多糖是由 N-乙酰-β-D-葡糖胺（GlcNAc） 通过 β(1→ 4) 糖苷键 连接而成的无分支线型同多糖；它与纤维素在化学结构上的唯一差别，就是每个葡萄糖残基的 C2 羟基（-OH）被乙酰氨基（-NHCOCH₃）所取代。壳多糖经浓碱加热脱去乙酰基后得到壳聚糖（Chitosan，脱乙酰壳多糖），是天然界罕见的带正电荷（阳离子）的无毒碱性多糖，广泛用于生物医用敷料与水处理。
3. 右旋糖酐（Dextran，又称葡聚糖）——从牙菌斑到凝胶层析柱 Sephadex：
   酵母和细菌的胞外贮存同多糖。右旋糖酐以 α(1→ 6) 糖苷键构成主链，并带有 α(1→ 3)（及少量 α(1→ 2) 或 α(1→ 4)）分支。口腔突变链球菌利用蔗糖合成黏稠的右旋糖酐，是形成牙菌斑（Dental plaque）和龋齿的罪魁祸首；而在生化实验室中，将右旋糖酐用表氯醇人工交联成网状珠体，就是大名鼎鼎的交联葡聚糖凝胶（Sephadex），广泛用于体积排阻层析（凝胶过滤层析）纯化蛋白质！

三、多糖动员的双轨制智慧：胞内糖原“加磷酸解” vs 消化道淀粉“水解”

现在，假设你正在操场上百米冲刺，或者刚刚吃完一碗热腾腾的土豆泥。你的身体需要从多糖（胞内的肌糖原/肝糖原，或消化道里的淀粉）中拆下葡萄糖单体送进糖酵解产能。但大自然在这里展示了一个令人拍案叫绝的双轨制设计——

请仔细观察上图对比的两条多糖拆解路线：

• 在消化道腔（胞外）拆解饮食淀粉——采用“水解（Hydrolysis）”：
  唾液和胰液分泌的 α-淀粉酶以水分子（H_2O）作为亲核试剂，切开淀粉的 α(1→ 4) 糖苷键，最终在肠刷状缘生成不带电荷的游离 D-葡萄糖（Free D-glucose）。为什么要生成游离葡萄糖？因为消化道腔在拓扑学上属于“体外”，分解出的单糖必须穿过小肠黏膜上皮细胞膜并进入血液循环（经 SGLT1 和 GLUT 转运蛋白）才能送往全身！如果在这里挂上带负电的磷酸基团，糖分子就会被挡在肠腔外无法高效跨膜转运。
• 在细胞质（胞内）动员内源糖原——采用“加磷酸解（Phosphorolysis）”：
  当肝细胞或肌细胞在胞质内拆解自己的糖原颗粒时，糖原磷酸化酶（Glycogen phosphorylase，辅基为维生素 B₆ 衍生物磷酸吡哆醛 PLP） 绝不用水分子去切，而是用胞质中丰富的无机磷酸（P_i，即 HPO₄^2-）直接进攻 α(1→ 4) 糖苷键的 C1 碳原子，发生加磷酸解作用（Phosphorolysis）：
  糖原_{(n 残基)} + P_i ──(糖原磷酸化酶)──► 糖原_{(n-1 残基)} + α-D-葡萄糖-1-磷酸（G-1-P）
  生成的 G-1-P 随后在磷酸葡糖变位酶（Phosphoglucomutase）催化下，不消耗任何 ATP，直接转变为糖酵解的第二步底物 6-磷酸葡萄糖（G-6-P）！（注：磷酸化酶切到距 α(1→ 6) 分支点前 4 个残基处会因位阻停下，再由具有“寡糖转移酶 + α-1,6-糖苷酶”双功能的脱支酶将其中 3 个残基转移到主链，并把分支点那 1 个残基水解为游离葡萄糖，因此糖原降解产物中约 90% 是 G-1-P，仅约 10% 是游离葡萄糖）。

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🎧 MIT 7.05 课堂原声英文实录对照（本节关联 1 讲 · 完整逐段实录）点击展开 / 收起 ▾

🎬 MIT 7.05 Lec 19 · Intro to Metabolism, Polysaccharides & Bioenergetics⏱ ~50:00

~00:00MITOCW | 19. Introduction Metabolism/Polysaccharides/Bioenergetics/Intro Pathways

~00:01MATTHEW

~00:02OK. So welcome back, everybody, to 705. I know this has been a really challenging semester for all of us. I want

~00:09VANDER

~00:09to particularly acknowledge for the seniors out there the tragedy of missing the end of your senior year.

~00:15HEIDEN:

~00:15Hopefully us, MIT can somehow make it up to you. I can say for my part, we will do our best to teach you biochemistry remotely. And just as a couple logistics, a reminder, I'm Matt Vander Heiden. I'm going to be covering the remaining part of 705. For the rest of the semester, we get to focus on biochemistry of metabolism. We're going to learn what metabolism is, why it's important. And basically, this can be quite a polarizing topic. In my experience, people tend to really love or really hate metabolism. It's interesting, because there are some people out there who pretend to hate it, but actually deep down really love it. Hopefully I will inspire you over the rest of the semester to at least have some appreciation for why metabolism is important. And regardless, for those of you who want to go to med school, this is a favorite topic of MCAT Exams. And also you will see that it will come up in many different areas of biology, regardless of whether you go to med school or not. So if you do anything further in biology, hopefully you'll find this material somewhat useful. OK, so getting into what metabolism is. So I think it's good to start with a textbook definition of metabolism. And so metabolism is the chemical reactions that all cells and organisms use to do two things. One, extract energy from the environment. And two, synthesize the macromolecules that make up all life. So really, what metabolism is then is the chemistry that makes life possible. And so understanding metabolism is really something that will help you better understand your food. There's lots of material out there in the popular press about what's healthy and what's not. At least this will allow you to form some of your own opinions about those claims, and how they relate to what really goes on in our cells and bodies. There's lots of implications for medicine, very relevant to agriculture, the energy sector, biofuels come from. And so metabolism really matters to tackle many of the challenges that face us in society. Now as a topic, this can be very daunting. As shown here on this image, we have hundreds, this is a typical metabolic pathway chart that is hanging in the walls of my office, many other laboratories and academic places around the world. If you look at this chart, it's filled with hundreds of enzymes, complex pathways. Our goal here is not to memorize this chart. You can always look up details of any pathway or reaction that you want. The goal here really is to see beyond this complexity. I want you to appreciate why metabolism works the way it does, why this chart is organized as it is. Because what we will find is that this chart is really variations on relatively few reactions. It basically is life repurposing similar chemistry to do many different things.

~02:53I think there is a beauty in how life can use these reactions to get such a diversity of macromolecules, and enable cells to solve all kinds of different problems. You will see that all of the pathways really follow common principles. These are shared across all species and all forms of life. And so at the chemical level, all life is really related. And this is why it's so relevant to understanding many of the challenges in medicine, understanding ecology, evolution. And for you MIT students, you'll see it's also very critical for using engineering approaches based on biology to solve various problems in the world. OK. So now let's start getting into this a bit more. Now, the first topic that we're going to cover over the next several lectures really relates to coming back to sugars and carbohydrates, which I talked about and introduced in my previous lecture. And we're going to do this because sugars and carbohydrates are key energy transduction molecules and cells. And it really facilitates a discussion of the principles for how metabolism works. Now our goal here isn't to memorize pathways. We could read about the glycolytic pathway in an hour. I could draw it up here in a very short period of time. Many of you have probably already done this in high school. Our goal is instead to understand this pathway at a deeper level, really see why the breakdown of sugars works the way it does, how cells use this to release energy, and how that can be used to support other cell functions. And so before we get to that, I want to start by introducing just a couple high-level concepts about metabolism. And so really, based on our definition before, metabolism is really about two things. It's about making stuff, and we have a fancy term for that. That is called anabolism. And it's about breaking stuff down. And the term for that is called catabolism. And anabolism, building stuff-- that is, producing biomass, growing all of the material that's out there, anabolic steroids help you grow. This is anabolism to build stuff. That is one side of metabolism. The other side is breaking stuff down, catabolism. That is, breaking down our food, eating food. Digesting our food is a way to get energy. Now of course, to reproduce, life has to build more cells. You need more stuff. You can't create something from nothing, and so that requires energy input. Anabolism typically requires energy input. Whereas that energy has to come from somewhere. And that's where catabolism comes from. We have to break down food. And ultimately, catabolism is the source for biological energy for many different systems. Now, some of this is actually somewhat intuitive. We all learned as little kids that we need to eat food if we're going to grow up and be big and strong. You also know that if you're going to run a race, you've got to eat a bunch of food, you need that energy to fuel your activity. But maybe what's less intuitive to many of you is we also need energy to sustain life, even if we're doing absolutely nothing. OK, adults, sit on the couch all day, hopefully they're not growing. But they'll still starve if they don't constantly eat some food, even if they're inactive. And so why is that the case? Well, hopefully some of you recall the second law of thermodynamics. So what is the second law of thermodynamics? It's, in effect, the entropy of the universe is increasing. That the universe continually tends towards disorder.

~06:05Life is exactly the opposite of this. Life is actually maintaining extreme order in the face of the second law. And so all life must constantly battle entropy. And many have described life at the very highest level as being really the ability to maintain order, to fight entropy, which of course requires constant energy input. And metabolism is the process that all cells use to do this. Now for humans, I think we all know we need to eat and breathe in order to stay alive. For you future MDs out there, what happens if your heart stops, or you stop breathing? Well, of course, you die in a matter of several minutes. Why is that? Well, that's because every cell in your body has to do constant catabolism in order to derive energy, in order to remain viable. This means they need constant food, and particularly oxygen delivery. And if you don't get those things, metabolism can no longer work, and the cells die. And so energy is sort of this mystical concept that we like to talk about. And so before we delve a little bit deeper into what I mean by biological energy, what it is, why we have to constantly do catabolism to maintain it, I want to go back and say just a little bit more about carbohydrates and finish our discussion there, and talk about carbohydrates and sugar polymers, polysaccharides, because this has some additional properties I didn't have time to cover in my prior lecture that is important to understand how it allows these molecules to store energy in a very efficient way. And it will also enable us to talk a little bit about how carbohydrates can also be used as structural molecules for lots of different organisms. All right, so a little diversion to discuss carbohydrates and polysaccharides. And so if you recall from my prior lecture, sugars that are greater than four to five carbons can exist either in open chain or ring forms, furanose or pyranose rings. I'll just remind you by redrawing up here, glucose. So this is D glucose, drawn in the open chain form. Remember this can form a ring. OK. So it's a reminder, carbon 1, 2, 3, 4, 5, 6. If we have this hydroxyl group from carbon 5, form a hemiacetal bond with the aldehyde at carbon 1, you get this ring structure. This would be alpha or beta, depending on whether the hydroxyl group here at carbon 1 is pointing up or pointing down. D-glucopyranose. So this is review of what we already talked about last time. Now, what I want to talk about is that if we form a disaccharide or a polysaccharide-- that is, begin to make sugar bonds, and if we do that in a way that ties up this hemiacetal or hemiketal bond, this then prevents the ring from opening and gaining access to that carbonyl, that aldehyde carbonyl, as this moves between the open chain and the ring form. And so a good example of this is the disaccharide sucrose. So sucrose is a disaccharide of glucose plus galactose. And here is what this disaccharide looks like. OK, so you have here, glucose in the alpha-D-pyranose form. Here's fructose in the beta, because the OH group is pointing out. Fructofuranose form. And so the formal name for this molecule, sucrose, would be alpha-D-glucopyranosyl 1-2, because we're going from carbon 1 of glucose to carbon 2, there's 1, 2, 3, 4, 5, 6 of fructose. Beta-D-fructofuranose. Sucrose or alphaD-glucopyranosyl 1-2 beta-D-fructofuranose.

~09:14OK, so this disaccharide forms a bond between carbon 1 and carbon 2. That would be the aldehyde in glucose, or the ketone tied up in the hemiacetal or the hemiketal bond of these two molecules. And so there's no way that either of these sugars can access the open chain form, without breaking this O-glycosidic bond. And that's one reason why sucrose is a very good storage molecule for carbohydrates, because without breaking that bond, you prevent access to gaining these reactive aldehyde or ketone bonds, that could react with some other molecule in the cell. Now, I mention this because whether or not a particular sugar is enabled to get access to this reactive aldehyde or ketone really forms the basis for a classic sugar detection lab test. And this is relevant to discuss this lab test because it explains some nomenclature that is still used, and in fact, we will continue to use a bit as we discuss some aspects of sugar metabolism. So what this test is, is basically, is that if you heat the sugar with copper, which is blue, and that copper can gain access to a free carbonyl--that is, if the sugar can access the open chain form to expose the aldehyde or a ketone, that copper can then become reduced to copper plus. That changes the color from blue to red. And so if you reduce the copper, you oxidize the bond there. And this test turns positive if there is a reducing sugar, is what it's called. So a sugar that reduces the copper from the blue 2 plus state to the red plus state is a positive test. And so basically, a reducing sugar is any sugar with an ability to access an open chain form. Because that will provide the free aldehyde or ketone to reduce the copper in this test. So this term, reducing sugar, obviously applies to all monosaccharides, because every single one of them has an aldehyde or a ketone, and can access the open chain form. But it will only apply to some disaccharides or polysaccharides. So let's come back to sucrose up there. So is sucrose a reducing sugar? No, it's not, because there is no way that you can access a free aldehyde or a ketone in sucrose, because it's tied up in that O-glycosidic bond between carbon 1 of glucose and carbon 2 of fructose. And so you need to break that bond to give them monosaccharide subunits in order to access the open chain form. However, let's give an example of a disaccharide that is a reducing sugar. And that's the disaccharide maltose. So maltose is basically two glucose molecules. And those two glucose molecules are like this. So this has a O-glycosidic bond between carbon 1 of this glucose, and carbon 4 of that glucose. And so formally, this would be alpha-D-glucopyranosyl 1-4 alpha or beta. If it's pointing down, it's alpha, pointing up, it's beta. Dglucopyranose. So maltose or alpha-D-glucopyranosyl 1-4 alpha or beta D-glucopyranose is a reducing sugar, because this sugar could access the open chain form. You could open up this carbonyl and expose the free aldehyde at position 1. Now, we could also refer to this sugar as having two different ends. We can refer to this end as being the nonreducing end, and this end as being the reducing end. Because of course, this bond, this aldehyde on the first glucose is tied up in this O-glycosidic bond, whereas the one on this one is not. So this end is non-reducing, and this end is reducing. Now this becomes much more relevant if we start talking about polymers. And so what starch is. So starch, what potatoes are made out of, is basically a polymer of maltose. Polymer of maltose molecules. So basically our glucose with 1-4 linkages.

~12:39OK, so I could draw that end, the reducing end of the polymer, in the alpha or beta. But this here, being the nonreducing end. Every other subunit is tied up in this 1-4 O-glycosidic bond. And so starch is really a polymer of glucose molecules with bonds between the 1 and the 4 position of each subunit. Now we will see later that we build and break down starch polymers from only one end, from the non-reducing end. And so having these terms, reducing and non-reducing, provides the term to specify different ends of a polymer. Reducing and non-reducing ends turns out to also be important for naming conventions for disaccharides or polysaccharides. And this becomes relevant if we consider the disaccharide lactose. So lactose is also a polymer of glucose plus galactose, except this polymer of glucose plus galactose, unlike sucrose, is different because it has a different linkage between the sucrose is a disaccharide of glucose plus fructose. Fructose, I wrote the name correctly. But lactose is a disaccharide between glucose and galactose. And so I'll draw it here. OK, so this is lactose. This is galactose here on the non-reducing end. So the formal name for this would be beta, because there's a beta linkage here between carbon 1 of this galactose and carbon 4 of this glucose. So be beta-D-galactopyranosyl 1-4. And this is glucose in the alpha or beta. D-glucopyranose. Lactose, or beta-Dgalactopyranosyl 1-4, alpha or beta, depending if I drew down, alpha, up, beta. D-glucopyranose. Now, this also has non-reducing and reducing ends. And by convention, you would name the sugar from the nonreducing end to the reducing end. Hence I put galactopyranosyl 1-4 alpha-D-glucopyranose, named them in that order. Now you'll note that lactose, unlike starch, unlike maltose, unlike sucrose, links these disaccharides with a beta linkage between this sugar and that sugar, where the other ones had alpha linkages. And structurally, this is very different. And I just want to point that out. So this here is basically alpha-D-glucopyranose. And so alpha, this is carbon 1. This is the hydroxyl pointing down. That's why it's alpha. If I make an O-glycosidic bond to another sugar, you can see that it points and creates this kinked structure. Now, if I were to make this beta, this hydroxyl rather than being here would be at this position on carbon 1. You can see that that now creates a very different geometry. Now you have a flat molecule, as opposed to a kinked molecule. Now, this has consequences, of course, for the enzymes that break down the sugars. Obviously, it's going to be a very different enzyme that breaks a bond in this orientation, versus a bond in that orientation. This has, basically implications for what enzymes we have. And so lactose, of course, is milk sugar. All mammals make milk, so make lactose, and break it down when we're babies. But most mammals tend to lose expression of the enzyme lactase that's able to break this 1-4 beta linkage as we age. And this is basically what accounts for lactose intolerance. Now, if you think about it, in the world, much of the world has issues with varying degrees of lactose intolerance in adulthood. The exception to this is often people of European ancestry. Why is this?

~15:36Well, Europeans historically drank milk well into adulthood. And so this selected for continuous lactase, the enzyme that wouldn't break that beta 1-4 linkage. The expression as people move into adulthood. And so it's an example of how cultural things, drinking milk into adulthood, really affected evolution, such that that subpopulation of people, people with that genetics to increase lactase expression, don't become lactose intolerant as they age. Now, this alpha versus beta linkage-- that is, whether or not you have this more kinked versus more flat structure-- also has a major effect on the structure of a polymer as well. And so if we look here at this starch polymer, it's basically this series of glucose molecules stuck together with alpha 1-4 linkages. So that's going to create a kinked structure. This is best shown here on this slide. So you can see this kinked structure. And so when you build this polymer, you're going to end up more with this helical structure in 3D space. That's a very efficient way to store glucose monomers in much less space than you would otherwise get with beta linkages. Now, it turns out that nature takes this a step further. And in addition to just having this starch polymer, it turns out sometimes you can add branches to the starch polymer to further increase the efficiency of energy storage. So if you have here a starch polymer, so let me just draw here a couple subunits. So this here is a starch polymer, glucose polymer with alpha 1-4 linkages that I can put an additional branch on this by adding on a linkage up here on the top. With an alpha 1-6 linkage. So the 1 position of here, to the 6 position of here, which has the effect of creating branches on this long polymer, such that you have a single reducing end, and lots of non-reducing ends. Each of these non-reducing ends has a polymer of alpha 1-4 linkages that would be stuck to the main chain polymer via this alpha 1-6 linkage. And this has a couple implications for storage. The first is, by only having one reducing end, it reduces maximally the exposure to a free aldehyde. And so that makes it a good storage sugar. And also as we will see, we're going to break down these sugar polymers biologically from the non-reducing ends, and build from those ends. And so there's lots of hooks or lots of places to either add or remove sugars from, which allows you to access carbohydrates quickly as needed. That's shown nicely here on this slide, better than I can draw it. So up here, you have your non-reducing end across the polymer. So this here would be starch with the alpha 1-4 linkages on the polymer, or reducing end on one side, non-reducing end on the other. You can then add branch points to that by making an O-glycosidic bond between carbon 1 down to carbon 6, creating a branch. And so you can see this helical branch structure will then form. That is both very efficient to pack lots of glucose into one place, and give lots of non-reducing ends with which to build and remove sugars from for biology to either store or quickly access sugar molecules as needed. Now, in plants, of course, potatoes use starch in a straight chain. We know that. However, plants also use this branched structure. This is a molecule called amylopectin. Amylopectin is better known in the food industry as Sure-Jell. It's the material that allows you to make jelly. It's the jelling agent in jelly. And effectively, this has every 24 to 30 units. There would be a branch with one of these alpha 1-6 linkages to give you this multi-branched structure.

~18:57Now, animals don't make starch. Animals don't make amylopectin. But animals make another molecule called glycogen. Glycogen, also a sugar polymer, exactly like amylopectin. Alpha 1-4 linkages with the alpha 1-6 to create branch points. Just like amylopectin, except glycogen has even more branches with a branch every 8 to 12 glucose units. And so, this is a very complex structure for both plants and animals to quickly store and quickly release glucose molecules when they're needed. Now, this is in contrast to a glucose polymer that can be done that has a very different 3D structure. So what if we take starch, and rather than have these alpha 1-4 linkages, but instead replace these with beta 1-4 linkages? OK, so now beta linkages. Looks simple enough, but changing the geometry from the alpha to the beta bond removes the kink. Now it's much more of a flat structure. Turns out this is, shown on an image here. So here's a beta linkage, and suddenly you have this flat structure. This is the same polymer as starch chemically, but has a different linkage, the beta 1-4 linkage. And that polymer with the beta 1-4 linkage is cellulose. And cellulose is, of course, what wood is made out of. And so wood and potatoes, cellulose and starch, exactly the same polymer. Same number of calories, if you could access the glucose units. But the alpha and beta bonds make them very different. Obviously, wood and cellulose is a very good structural polymer for plants. We build houses out of it. We don't build houses out of potatoes, but we eat a lot of potatoes. So equally good molecule for plants to store lots of food for energy. Turns out wood is a great source of energy too. You just need to have an enzyme that can break the beta 1-4 bond. Termites have symbiotic microbes that allow them to do this, and this is why termites can eat wood, a lot of energy tied up in wood. Now, we lack the time to go into the details about how this relates to other structural carbohydrates. But in general, structural carbohydrate or carbohydrate-like molecules form polymers of sugar or related molecules, also with these beta linkages. And a good example is chitin, so the material in the insect shells, is basically a sugar-like molecule that's a polymer with a beta linkage in it. Same thing with cartilage in humans and animals. And you can look up the details of what these things look like, of course, if you're interested. Chitin and cartilage aren't made of true polysaccharide polymers, but they're very related to polysaccharides. And they really illustrate how nature can take carbohydrate chemistry and repurpose it to basically act as an energy source, but also build all kinds of structural molecules that are really useful in biology. All right, now I want to go back to, here's ways that one can store and use carbohydrates in interesting ways. But now I want to come back to them as energy sources, and assuming you can access the glucose and whatever polymer it's present in, how can you actually metabolize it to provide energy in a way that sustains life? And this means we're going to turn to another topic, a topic that's typically referred to as bioenergetics. Bioenergetics, which is really the discussion of how energy is transduced in biological systems.

~21:58I'm going to introduce this topic today, we're going to revisit it throughout the course. But it's really important to ask this question. We really want to consider, what do we mean when we say biological energy? What is biological energy? Well, someone probably made you memorize in high school, and often you were thinking, oh, it's just ATP. And certainly, ATP, adenosine triphosphate, is a very important molecule for biological energy transduction. But I want to actually explore why ATP is actually useful. And if ATP is energy, why don't we just eat all kinds of ATP? I can say for a fact that absolutely none of you had ATP today for breakfast. No one sells ATP as an energy booster. And so if ATP is such a great energy source, why don't we just eat it? And to really understand ATP, how it works, what biological energy really is, we really need to revisit some very basic topics in thermodynamics.

~22:50Now, thermodynamics strikes fear into the hearts of students everywhere. You can get details of thermodynamics and the theory around these things in other courses. That's not really what we're going to try to accomplish here in 705. Our goal here is really a practical understanding of how thermodynamics applies to biology and metabolism. And we need to go into this, because it's key to understanding why we store energy, go through all this trouble to store energy as these carbohydrate polymers to begin with. Why we eat potatoes and not ATP, as well as why ATP is actually useful to cells. And so let's take a step back and just think about it. Let's think about wood. I just told you, wood is a polymer of glucose molecules with beta 1-4 linkages. How can we, forget as organisms, as cells. But how can you release energy from wood, to do other kinds of work? Well, we can burn it. And so what's the chemistry of burning wood? Well, it's a carbohydrate polymer. So it has this CnH2n formula. If we combine that carbohydrate with oxygen, it releases CO2 and water, plus some light, plus heat. This light and heat is energy, because we can use it to do work. Boil water, turn a turbine, make electricity. Whatever. Warm ourselves by the fire. Use the light to do some other things. And so this burning of wood is certainly release of energy. Now, we lack the enzymes to do this reaction in wood, because we can't break that beta 1-4 linkage. But we certainly can break that alpha 1-4 linkage in starch from potatoes. And we use the exact same chemistry to burn that glucose and release energy. Except the difference is that if I burn wood, I do this all in one step. But life is a much better engineer than that. It basically, rather than releasing all of that energy in one step, it really releases it stepwise in a manner that's actually useful to cells. But it's exactly the same release of energy. And so what life is, or extracting biological energy is, is it's really the ability to do stepwise oxidation of glucose or other carbon. I'll write glucose for now. To get energy. So we burn wood, that's favorable, it releases energy. If we do stepwise oxidation, we also can release energy as well. Same reaction, also releases energy.

~24:59How much energy do we get from releasing us burning glucose, as you do and burning it in a fire? Well, it has to be exactly the same amount, because it's exactly the same reaction. So we burn wood, we get light and heat. Cells burn glucose, it releases the exact same amount of energy. It just does so in a way that allows the cells to do biologically useful things. What are those? Well, it could be heat. All of us maintain temperature. So heat is a conversion of energy that of course, is useful to biology. But it can do other things, too. It can allow cells to move. It can allow them to do any reaction to fight that entropy that allows life to exist. And so all things, including ourselves, have to follow the same laws of thermodynamics. Life is not special in that regard. And so burning wood is favorable, because it increases the entropy of the universe. You're breaking up this polymer into a bunch of monomers. That's going to be spontaneous. That is in accordance with the second law of thermodynamics. True if cells burn it as well. But remember, the second law says that net entropy of the universe must always increase. And so if we're going to do something that's not energetically favorable, like build a glucose polymer, we obviously have to put energy into it. And to do so, the energy that's released must be greater than the energy that we actually store. And so this is actually a really key point, because anything that we do that requires energy input for a cell to carry out that process requires a source of energy release somewhere else that is equivalent or greater than what is actually put in. And so cells need energy, because they do lots of thermodynamically unfavorable things, fighting that entropy of the universe. And all of those processes must come from the release of energy elsewhere, like the burning of carbohydrate. And so that constant energy input is at the very highest level, why we need to do constant metabolism in order to maintain order and survive as organisms. Now this, of course, comes from things like burning of carbohydrate. But of course, that carbohydrate has to come from somewhere. And so ultimately, you have to have an external source. That external source is of course, the sun. Photosynthetic organisms can use light energy from the sun to do exactly the same things. Build those excess polymers, and that's why we as animals or anything that eats other animals as a way to live is ultimately depending on eating photosynthetic organisms, because photosynthetic organisms do this by harvesting energy of the sun. Now that's all very high level. But I want to come back to the specifics of metabolism, ultimately, and understand those as how they relate to enzymes' reactions and pathways. That is, how do you get that complex series of reactions that make life possible, and ultimately still be guided by these exact same principles? In other words, how do we couple the energy releasing reactions, like burning glucose, in a way that obeys the laws of thermodynamics and operates under biologically acceptable conditions in order to do things that are biologically unfavorable. That's really what bioenergetics is. And so the next thing we want to consider then, if we want to understand this, is really, let's ask the question. What determines if a specific reaction occurs? That is, what determines if something like burning wood actually spontaneously happens?

~28:08If you light wood on fire, it will burn. It does so every single time, everyone has had that experience. But no one has ever seen CO2 and water spontaneously come together and form a tree. That doesn't happen. So what determines whether you burn wood and that is favorable, versus why it is that CO2 and water don't come together and spontaneously form a tree? Do not think that this is determined entirely by enzymes. Enzymes are catalysts. Enzymes are the spark that makes the burning of wood possible. But remember, enzymes cannot change thermodynamics. Enzymes only change the rate at which a reaction happens. It does not change the equilibrium. That tree wants to, if given the catalyst, form CO2 and water. That reaction is spontaneous because it is thermodynamically favorable. That tree will not take CO2 and water and spontaneously reform a tree. And there's no amount of enzyme that will make that happen all by itself. And so remember that. If you have any reaction between A and B, there is some equilibrium, as defined by the chemical properties of A and B, such that the equilibrium lies far to one side or the other. And that equilibrium is determined by thermodynamics. It is not determined by enzymes. I don't care how much enzyme I add. If the equilibrium lies towards A as I draw it, I can add all the enzyme I want, and I can never create more B. And so, this is super important, and is actually a point that is gotten wrong by lots of biologists. They think, oh, this enzyme is expressed. Therefore, this pathway must be happening, or this reaction must be happening faster. You need an enzyme to catalyze a reaction. So a reaction may not occur, your wood may not burn unless you give it a spark, a catalyst to actually make it happen. But you cannot add a catalyst to fight thermodynamics. You cannot take that CO2 and water and turn it back into a tree. And the same thing is true. You can add all the enzyme in the world you want, and all it will do is help A and B establish the equilibrium that is defined by the thermodynamics of the relationship between A and B. Now, what determines the equilibrium between any two species? That's a topic for another class, why a species is favored to be one side or another. However, a useful tool to think about this in biochemistry and quantify this for any pair of reactions is something you've probably learned about before. It's something called the Gibbs free energy. Gibbs free energy, or delta G. Delta G is related to the way we'll talk about the equilibrium constant. And it's basically a term that we can use to specify if a reaction is favorable or not. And so hopefully you learned in an introductory class that if delta G is less than 0, our reaction is spontaneous. If delta G equals 0, our reaction is that equilibrium. If delta G is greater than 0, our reaction is not spontaneous. All right, if we take our log and ask, what is delta G to turn that log into CO2 plus water, it is less than 0, because it is very spontaneous. We take a pile of ashes and ask what delta G is to recreate the log. It's greater than 0, because it ain't going to happen. And if it's at some equilibrium, delta G is equal to 0. Now, delta G depends entirely on conditions, as we will see in a minute. And because of that, this means that there can be conditions where absolutely any reaction can be favorable if the conditions are right. And we'll see that this becomes very relevant to understand how metabolism works. And so let's go back and just now consider some generic reaction, A and B.

~31:37So at equilibrium, what happens? So at equilibrium, the concentration of A and the concentration of B are not changing. Delta G equals 0. These two things are at equilibrium, whatever that equilibrium is. A and B are not changing in concentration. Now if I come and I add more A to this side of the equation, what does that mean? Well, you know from Le Chatelier's principle that that's going to favor production of B. We have shifted things now out of equilibrium. So if I add A, that leads the production of B to re-establish this equilibrium. That means that delta G from A to B is less than 0 until I've reestablished equilibrium, and now delta G is back to 0 again. Now, what happens if I add B? Well, if I add B, now I'm going to produce some A. So add B. That leads the production of A, until I reestablish that equilibrium. So delta G, in that case, to go from B back to A. The reverse reaction is less than 0. Or I could also say that delta G, to go to A to B here, because that's not going to happen. I just added more B. I'm not going to suddenly create more B from A. So here now, delta G is greater than 0. Now note when I did this, I don't actually have to specify how much A or how much B I've added. The equilibrium depends on the ratio of B to A. Not the absolute concentration of either species. And so having a way to think about this equilibrium, it turns out, is also useful for biological systems to divine some delta G that is helpful to relate to this equilibrium constant. And that is this concept of delta G 0 prime. A standard free energy, which for biological systems, relates to the equilibrium constant at 25 degrees, or the pH of 7 and 1 atmosphere. Pretty typical biological conditions. And this here is basically related to the equilibrium constant. And we can calculate the actual delta G by the following formula. Delta G equals delta G 0 prime plus RT times the log of the products over the reactant. So this would be drawn for the reaction A to B. So the reaction A to B is related to delta G 0 prime, which is related to the equilibrium constant. I'll tell you how in the second. Plus R is the gas constant, T is the temperature in Kelvin times the natural log of the ratio of products, B over reactants, A. This tells you whether or not at specific concentrations of B and A, that is a specific ratio of B and A, whether it is favorable to go from A to B, delta G less than 0, or whether it is favorable to go from B to A, delta G greater than 0. And so this should begin to make it clear that whether or not a specific reaction occurs is affected by the actual conditions present. And you can calculate that based on this relationship. And so if I specify concentrations of A and B, as well as delta G 0 prime, I can know, at those concentrations, which direction of the reaction is favored. Now of course, my drawing A to B is entirely arbitrary. I could equally draw it B to A. And if I did that, so this is delta G 0 prime, A to B, all I do is flip the signs. So delta G equals delta G 0 prime. The negative of A to B is equal to the positive of delta G 0 prime from B to A, because the direction is arbitrary. Plus RT log in this case. Now I have A is my product, and B is my reactant. If I just flip the sign, flip that ratio, it's just going to change the sign of my product and give me exactly the same result with the opposite sign, which makes sense. What's favorable in one direction is not favorable in the other direction, and vice versa.

~35:17Now, I can also set delta G equals 0. That's equilibrium. If I do that, then delta G 0 prime equals negative RT. So this would be for A to B. I'll use the top example. So delta G equals 0. Then I have delta G 0 prime equals RT times log B over A. So if I know two things are at equilibrium, I can calculate delta G 0 prime, and know what the equilibrium constant is. And so it follows from this then that if delta G 0 prime is negative, that means B is favored over A at equilibrium. And if delta G 0 prime is positive, that means A is favored over B at equilibrium.

~35:55And so delta G 0 prime really is a convenient way to look at a reaction, and know which direction the equilibrium lies. So A and B, delta G 0 prime is negative, equilibrium lies to B. Between A and B, if delta G 0 prime is positive, the equilibrium lies towards A. But the key concept is that for any specific reaction, any specific conditions of A and B, whether or not A is converted to B will be defined by the equilibrium constant. But it will also be defined about what delta is under those conditions. So to be clear, delta G will depend on conditions, because I'll write it again, delta G equals delta G 0 prime plus RT times the log for the reaction, A goes to B. So products over reactants. And so whether or not A is converted to B will be a property of the equilibrium constant, plus the concentration of B and the concentration of A, that ratio of concentrations at the conditions present. And if in that calculation, delta G is less than 0, that reaction is spontaneous. If delta G is greater than 0, that reaction is not spontaneous. Or to put it another way, if delta G is less than 0 under those conditions, energy is released. Or if delta G is greater than 0, that reaction will not occur without energy input. If I burn wood, energy is released. But there's no way I can reassemble that log without some kind of energy input. And really getting this concept is central to understanding metabolism, as well as what biological energy means. And hopefully, what is now apparent, based on what I just said, is that what really determines delta G is two things. Of course it's the equilibrium constant. But in essence, delta G for any reaction is proportional to the ratio of the reactants over the products. And that I can come up, at least in theory, with any relationship, any ratio of reactants to products to make this favorable. At some tiny, tiny concentration, if I have nothing but CO2 and water, and infinite time and infinite catalysis, a tiny bit of wood could spontaneously form. And so life really creates the conditions that allow that, and select for that to happen, and of course, add energy input so it can actually happen more. And so this should be very clear to you that absolutely no reaction is irreversible. I don't care what your textbook says. Lots of sources will discuss irreversible reactions. I'll talk about irreversible reactions later in the course. But what we mean when we say the word irreversible reaction is these are under conditions that are found in cells and in nature. And I stress this because if we want to understand the energetics for how pathways work, we have to appreciate that life can create conditions to do unfavorable things, because that's the only way that pathways can work.

~38:33All right, what what do I mean by that? Well, suppose again, we'll come back to our reaction. A goes to B. Suppose I need to build a pathway where I need to convert A to B, because B is actually useful for some purpose that I need to do in a cell. However, what if this is at least by equilibrium, unfavorable? What if the equilibrium lies towards A? That means delta G 0 prime is positive, is greater than 0, for A to B. What does that mean? Delta G 0 prime greater than 0, for the reaction A to B? That means the equilibrium lies towards A. And so there is no amount of enzyme I can add that will allow me to net convert A to B, because the equilibrium lies far towards A. However, whether or not a cell is able to build a pathway, where it converts A to B depends on delta G, not the equilibrium constant for that specific reaction. So how can I turn A into B? Well, I could turn A into B if I keep the concentration of B low enough that delta G, not delta G 0 prime, that's the equilibrium constant, but delta G favors A to B conversion? That is, delta G for A to B is less than 0. How can I do that? Well, remember delta G equals delta G 0 prime plus RT log of B over A. So this term is positive, because I told you, the equilibrium lies towards A. So I need a concentration, a ratio of B to A, such that B is low enough that this term is more negative than that term is positive. And if that's the case, delta G will be less than 0, and I can net move that forward. So how can I do that? How can I keep the concentration of B low? Well, I can build a pathway that consumes B in a way to keep it low, which favors A to B conversion. In other words, I can build a pathway, A goes to B goes to C. So even here, where delta G 0 prime is greater than 0, if delta G 0 prime for this reaction, B to A, is much less than 0, such that equilibriums strongly favor C, that means my unfavorable equilibrium here can be overcome by the very favorable equilibrium of B to C. In essence, I can use B to C to pull A to B, such that it actually happens. Produce this intermediate that's useful on the path to making C. This strategy is used in lots of metabolic pathways. And it's useful to generate lots of chemically useful intermediates. Now note that this only will work if conversion of A to C is favorable. That is, if delta G 0 prime is less than 0. I can only pull this reaction if delta G 0 prime is less than 0. That is, if equilibrium favors A to C, I can build a pathway this way to do something unfavorable on the way to making C. This brings up another key point, in that everything that we discuss, relationship between any two metabolites in any single reaction, also must be true for entire pathways. So I have a three-step pathway from A to C. Whether I turn A into C in one step, or turn A into C in multiple steps, the free energy is exactly the same. If I burn wood by lighting it on fire, it releases the same amount of energy than if a termite has an enzyme that breaks that beta 1-4 linkage in the wood, and can burn that glucose stepwise through metabolism, the exact same amount of energy is released. Glucose on one side, CO2 and water on the other side, exactly the same amount of energy released, whether I do it in one step, or I do it in multiple steps. The equilibrium constant between those has to be the same, and the delta G has to be the same between all of it.

~42:13And so it should become apparent, then, that A to C has to be favorable if I'm going to use this trick of keeping product low in order to pull an otherwise unfavorable reaction forward. In other words, there's no way for me to keep B low and net convert A to B all on its own. That will not work. And it's these unfavorable reactions that ultimately then require energy input. And this is where ATP now starts becoming useful for cells. OK, so now I want to talk a little bit about how ATP provides energy that allows otherwise unfavorable reactions to occur. And ATP is useful because delta G applies to sets of reactions in the same way it applies to single reactions. That is, it applies to whole systems. All reactions are a series of reactions, whether they happen alone or coupled together. All have to follow the same rules. So now let's make our reaction more complicated. Let's say A plus B goes to C plus D, where this is really two reactions coupled together. A being turned into C, and B being turned into D. All right, so will this reaction happen? Can I turn A plus B into C plus D? Well, how do I know? Well, it's going to be, got to calculate delta G. And so it's going to be delta G 0 prime for A going to C, plus delta G 0 prime for B going to D. So those will relate to what's the equilibrium constant, the equilibrium relationship between A and C and B and D. If it's less than 0, equilibrium will favor to the right. If it's greater than 0, equilibrium will favor to the left of A and C and B and D. Add them together, that gives me my overall delta G 0 prime for those two reactions. Plus RT times the log of the products. So that's C and D over A and B. Products over the reactants. So if I take an unfavorable reaction-- say, A to C, if delta G 0 prime is greater than 0, so it would favor A, and I couple it to another reaction, B to D that's very favorable, because I add these two terms together. I can now take something where the equilibrium would be unfavorable, and make it favorable. However, whether or not this actually happens still depends on this ratio of products to reactants. So it still depends on the ratio of C to A, and D to B in our hypothetical thing here. So to know if a specific reaction will actually occur, we have to take into account both the sum of the delta G primes related to the equilibrium constant, and the actual ratios that are present in cells. And so the net effect is that I can couple more favorable reactions to make otherwise unfavorable reactions now become favorable. That is, have energy input. But it still depends on conditions. And so let's think a little bit about the polymer synthesis that we learned from Professor Yaffe. And so in that process, what did we do? We made several polymers, and you learned about the chemistry to do this. We made DNA, we made RNA, and we made protein. These are polymers of nucleic acids. Protein is a polymer of amino acids. All of these fight entropy, right? We're building polymers, not breaking them down. And all of them were synthesized using reactions that hydrolyzed ATP. In fact, those reactions actually hydrolyzed ATP in the following way. And every single one of those cases, if you look back, what you will see is that they all had steps where ATP was taken to AMP plus 2 inorganic phosphates. This reaction is very favorable. In fact, it's two reactions. It's really ATP goes to AMP plus pyrophosphate.

~45:39And then that pyrophosphate goes to 2 inorganic phosphates. It turns out that delta G 0 prime is less than 0 for both of these reactions. That means the equilibrium lies towards AMP plus pyrophosphate. And here, the equilibrium lies towards 2 inorganic phosphates. And so there's actually two tricks here by metabolism. One is, it's two very favorable reactions that we're coupled to do something unfavorable, build a polymer. And use the trick of keeping pyrophosphate low by coupling it to a downstream, very favorable reaction, which even further pulls this reaction, and is why ATP hydrolysis here becomes so useful as a way to build these polymers. Let's just go quickly and add a few numbers to show exactly what I mean by how ATP hydrolysis can be useful in this setting. And so, let's do something a little simpler. Let's go to ATP plus ADP plus to inorganic phosphate. So let's give this some numbers, delta G 0 prime in this case is equal to negative 7.5 kcals per mole. All right, what does this mean? Delta G 0 prime is related to the equilibrium constant. It's less than 0, which means the equilibrium lies towards ADP plus phosphate. Now let's see how this actually helps by considering what the first step in glucose metabolism by cells is. So the first step cells do in glucose metabolism is add an inorganic phosphate group to glucose. So it's this reaction, glucose plus inorganic phosphate goes to glucose phosphate. Just a quick note on shorthand that I will use in this course. So PI, of course, is inorganic phosphate. That is this PO43 minus group. When it forms a phosphodiester bond, like to an alcohol and one of the glucose molecules, I'll draw this in a future lecture. I'll oftentimes indicate it by putting a circle around the phosphate. And so adding this phosphate to glucose is a step that traps that glucose in cells. For now, let's just consider this reaction. And so delta G 0 prime of this reaction is positive 3.3 kcals per mole. What does that mean? Delta G 0 prime relates to the equilibrium constant. It says the equilibrium constant lies here to the left, to the glucose plus phosphate side. So if we want to net trap glucose phosphate in cells, well, it's not going to happen spontaneously. You need some kind of energy input. That can come from ATP. And so if we couple those two reactions as follows. So we now have glucose plus ATP goes to glucose phosphate plus ADP. Now let's draw this out. Will this happen? So we can calculate delta G. Delta G equals delta G 0 prime. So there's two reactions happening here, glucose to glucose phosphate, and ATP to ADP. We know what delta G 0 prime is by adding those together. So it's 3.3 plus negative 7.5. That equals negative 4.2. What does that tell us? That says, now we've created conditions where the equilibrium constant lies towards the right, towards the glucose phosphate plus ADP side. But how much of this occurs or whether it occurs isn't just the property the equilibrium constant. It's also how much is there. And so that's RT times the log of the products, glucose phosphate.

~48:34And ADP over glucose. And ATP. And so, if this term is less than 4.2-- that is, if the ratio of glucose phosphate to glucose and ADP to ATP in this term ends up being less than positive 4.2, this reaction is spontaneous. It is favored. Lots of conditions where that would be the case, although if this term is 4.2 or greater, now it will no longer occur. How much ATP is needed? Well, it's hard to answer this question in absolute terms, because if there's a lot of ADP around, you need a lot more ATP for the ADP to be useful. And this is the problem with equating ATP with energy. The energy here is in the ratio of ATP to ADP, or ADP to ATP. And this is something that we we'll come back to in the next lecture, because in the end, this is what will determine if a reaction has happened. It is not the absolute concentration of ATP. It's the fact that ATP hydrolysis is favorable. But how favorable that is depends on the relevant concentrations of ATP or ADP. And relative concentrations are all that matters in thermodynamics. It is not absolute concentrations. It is the ATP-ADP ratio that is the correct way to think about energy. And this is true for any reaction with respect to energetics. If an ATP-ADP ratio exists, such that when coupled to a reaction, delta G is less than 0, then it becomes spontaneous. And this is how ATP provides energy. And we will come back to this more in the next lecture. Thank you.

1.4杂多糖与糖缀合物：从细菌铠甲、胞外基质水凝胶到细胞表面的“糖密码”

一、植物细胞壁基质与细菌细胞壁铠甲：溶菌酶与青霉素精准打击何处？

由两种或两种以上不同单糖（或单糖衍生物）聚合而成的多糖称为杂多糖（Heteropolysaccharides）。它们在植物、微生物和动物细胞外构建起了坚韧而富有弹性的支撑网络。

1. 植物与藻类细胞壁杂多糖：

• 果胶物质（Pectin）：存在于植物初生壁和胞间层，起黏合细胞的作用，主链是 α(1→ 4) 连接的聚 D-半乳糖醛酸（羧基部分甲酯化者称果胶，完全去甲酯化者称果胶酸；在食品工业中与支链淀粉同为果酱胶凝剂）。
• 半纤维素（Hemicellulose）：能被 15% NaOH 提取的植物细胞壁基质多糖总称（包括木聚糖 β-D-Xylp(1→ 4)、葡甘露聚糖等）。高频判断陷阱：半纤维素与纤维素虽然名字相近，但结构毫无血缘关系！纤维素是同多糖（纯葡萄糖），而半纤维素是含木糖、甘露糖、阿拉伯糖等多种单糖的杂多糖，绝不是纤维素合成或降解的中间产物！
• 琼脂（Agar）：从红藻提取的多糖混合物，其未分支主成分琼脂糖（Agarose）由 D-半乳糖与 3,6-脱水-L-半乳糖交替组成，加热溶解、冷却至 40～ 50^°C 形成惰性多孔凝胶，是微生物固体培养基、核酸琼脂糖凝胶电泳支持物及交联琼脂糖层析介质（Sepharose）的基石。

1. 细菌细胞壁的核心铠甲——肽聚糖（Peptidoglycan，又称黏肽）：
   为什么细菌在极低渗透压的纯水中不会像红细胞那样吸水胀破？因为细菌质膜外包裹着一层坚不可摧的刚性共价网状巨型袋——肽聚糖！

3D3D 糖类：杂多糖（植物壁、细菌肽聚糖与动物糖胺聚糖）交互查看果胶与半纤维素、细菌肽聚糖溶菌酶/青霉素靶点、G⁺ 磷壁酸 vs G⁻ 脂多糖 LPS 内毒素、糖胺聚糖（HA/KS/肝素三大特例）↗3D3D 酶机制：溶菌酶催化细菌肽聚糖 β(1→4) 糖苷键水解结合第 8 章溶菌酶 Glu35 + Asp52 酸碱与共价催化机制理解肽聚糖主链裂解↗

请对照上图，牢记肽聚糖的三层精密构造与两大抗菌武器靶点：

• ① 聚糖主链：由 N-乙酰-D-葡糖胺（GlcNAc） 和 N-乙酰胞壁酸（MurNAc） 通过 β(1→ 4) 糖苷键 严格交替连接成线型长链（注意：纤维素、几丁质、肽聚糖三大结构多糖的主链全都是扁平耐拉的 β(1→ 4) 糖苷键！）。
• ② 四肽侧链（含独特的 D-型氨基酸）：每个 MurNAc 残基 C3 位的 D-乳酸羧基上，共价连接着一条由 4 个氨基酸组成的短肽（如金黄色葡萄球菌中为 L-Ala—D-Glu—L-Lys—D-Ala）。为什么细菌要在四肽侧链里掺入罕见的 D-谷氨酸（D-Glu）和 D-丙氨酸（D-Ala）？ 因为人体和宿主体内的所有蛋白酶都只识别并切割由 L-型氨基酸组成的肽键！细菌巧妙掺入 D-型氨基酸，就给细胞壁穿上了“防宿主蛋白酶防弹衣”！
• ③ 肽交联桥（G⁺ 与 G^- 菌的差异）：在革兰氏阴性菌（G^-，如大肠杆菌）中，相邻聚糖链的四肽侧链直接通过肽键交联，肽聚糖层很薄（仅约 2 nm），但外侧还包有一层富含脂多糖（LPS）的外膜；而在革兰氏阳性菌（G⁺，如金黄色葡萄球菌）中，相邻四肽侧链之间由一条五聚甘氨酸桥（Gly₅，Pentaglycine bridge）在转肽酶催化下交联成致密的三维立体网格，肽聚糖层多达数十层（厚达 10～ 20 nm），并穿插有磷壁酸（Teichoic acid，由核糖醇或甘油与磷酸交替连接而成，为 G⁺ 菌特有抗原）。
• ④ 脂多糖（LPS，革兰氏阴性菌内毒素 Endotoxin）：位于 G^- 菌外膜外叶，由三部分组成——锚定于外膜的脂质 A（Lipid A，决定内毒素致热与休克毒性的核心部位）、核心寡糖以及暴露在最外侧的 O-特异侧链（O-抗原，有 O 链者菌落呈光滑 S 型，缺失者呈粗糙 R 型）。
• ⑤ 两大经典抗菌武器的精准打击位点（年年必考！）：
• 溶菌酶（Lysozyme，存在于眼泪、唾液、鼻涕和鸡蛋清中）：专门水解肽聚糖主链中 GlcNAc 与 MurNAc 之间的 β(1→ 4) 糖苷键，直接剪断聚糖骨架，使已经建好的细菌细胞壁彻底解体，细菌随即吸水渗透胀裂！
• 青霉素（Penicillin 等 β-内酰胺类抗生素）：青霉素在立体构象上高度模拟四肽侧链末端的 D-Ala-D-Ala 二肽，能专一结合并不可逆抑制催化肽交联桥形成的转肽酶（Transpeptidase），从而阻断新合成肽聚糖链之间的肽交联！
• 华农 2024 年判断题考点：青霉素只能抑制正在分裂繁殖的细菌合成新细胞壁（使新壁无法交联而胀破），而绝对不能破坏已经合成交联好的旧细胞壁（因此青霉素对处于静止期不分裂的细菌无效）；相反，溶菌酶可以直接拆毁已经形成的细胞壁 2024·判断！

二、动物胞外基质水凝胶：糖胺聚糖（GAG）与蛋白聚糖（Proteoglycan）

在动物组织中，细胞外面没有僵硬的细胞壁，取而代之的是充满弹性和抗压缓冲能力的细胞外基质（Extracellular matrix, ECM）。ECM 的核心水凝胶基质是由糖胺聚糖（Glycosaminoglycans, GAGs）与核心蛋白（Core protein）组装而成的蛋白聚糖（Proteoglycans）。

3D3D 糖类：杂多糖（植物壁、细菌肽聚糖与动物糖胺聚糖）交互查看果胶与半纤维素、细菌肽聚糖溶菌酶/青霉素靶点、G⁺ 磷壁酸 vs G⁻ 脂多糖 LPS 内毒素、糖胺聚糖（HA/KS/肝素三大特例）↗

1. 糖胺聚糖（GAGs，曾称黏多糖 Mucopolysaccharides）：

• 糖胺聚糖（Glycosaminoglycan, GAG）：又称黏多糖，是动物和细菌特有（植物中不存在）的一类不分支长链杂多糖，由重复的二糖单位 [己糖醛酸 → 己糖胺]_n 串联而成，多数带有大量硫酸酯基（-OSO₃^-）和羧基（-COO^-）。
• 其二糖单位中的己糖胺通常是 D-葡糖胺（GlcN）或 D-半乳糖胺（GalN）的乙酰化及硫酸化衍生物；己糖醛酸通常是 D-葡糖醛酸（GlcUA） 或其 C5 差向异构体 L-艾杜糖醛酸（IdoUA）。由于沿链密集分布着负电荷（-COO^- 和 -OSO₃^-），同种电荷相互排斥迫使 GAG 分子在溶液中呈充分伸展的棒状构象，并吸引海量水合阳离子（Na⁺）和水分子，形成可承受巨大机械挤压的弹性水凝胶。
• 六大糖胺聚糖必背特例（选择题最爱考“谁是例外”）：
• 透明质酸（Hyaluronic acid, HA）：由 [GlcUA(β1→ 3)GlcNAc] ──(β1→ 4)──► 重复多达数万次组成（相对分子质量数百万），存在于关节滑液、眼球玻璃体和软骨中，起润滑与防震作用（病原菌和精子分泌的透明质酸酶可水解它以利侵入）。透明质酸有两大独一无二的特例：① 它是唯一不发生硫酸化的糖胺聚糖；② 它是唯一不与蛋白质共价结合形成蛋白聚糖的糖胺聚糖（在软骨聚集体中仅作为中心主干，靠非共价键串联上百个蛋白聚糖单体）！
• 硫酸软骨素（Chondroitin sulfate, CS）与硫酸皮肤素（Dermatan sulfate, DS）：赋予软骨、肌腱、韧带和皮肤抗张强度（DS 中大量 GlcUA 差向异构化为 L-艾杜糖醛酸）。
• 硫酸角质素（Keratan sulfate, KS）：存在于角膜、软骨、毛发、指甲中。硫酸角质素是六大糖胺聚糖中唯一不含己糖醛酸的特例——它用 D-半乳糖（Gal） 取代了糖醛酸！
• 肝素（Heparin）与硫酸乙酰肝素（Heparan sulfate）：由肥大细胞合成并贮存于颗粒中的肝素，是 已知生物大分子中负电荷密度最高的物质！临床上广泛用作天然抗凝血剂，其机制是通过高密度负电荷与血浆中的抗凝血酶 Ⅲ（Antithrombin Ⅲ）结合并引发构象激活，使后者灭活凝血酶（凝血因子 Ⅱa）和因子 Ⅹa 的速率暴增上千倍！

1. 蛋白聚糖（Proteoglycan）vs 糖蛋白（Glycoprotein）——“看后两个字定本质！”：
   很多同学在考场上容易把“蛋白聚糖”和“糖蛋白”搞混。请记住一句秒杀口诀：中文偏正词组看后半截中心词——“蛋白聚糖”中心词是“聚糖”，本质是以糖为主；“糖蛋白”中心词是“蛋白”，本质是以蛋白质为主！

• 蛋白聚糖（Proteoglycan）：由一条或多条硫酸化糖胺聚糖长链通过共价键（常经 -GlcUA-Gal-Gal-Xyl-O-Ser 四糖接头）连接于核心蛋白（Core protein）丝氨酸残基（常位于 -Ser-Gly-X-Gly- 序列）上构成的巨型糖缀合物；按质量计糖多于蛋白（糖含量高达 95% 以上），是动物细胞外基质的主要结构与功能成分 2007·填空 2026·名词。

三、糖蛋白的糖肽键（N-连接 vs O-连接）与 ABO 血型“糖密码”

如果说多糖是细胞的燃料库和承重墙，那么共价连接在质膜表面蛋白质和脂质上的短分支寡糖链（Oligosaccharide chains），就是细胞对外亮出的“身份证”和“信息天线”！为什么寡糖链比多肽链或核酸链更适合当高密度信息载体？因为 4 个不同核苷酸或氨基酸只能形成 4⁴ = 256 种四聚体（肽键和磷酸二酯键只有唯一 1 种连法）；而 4 个不同单糖由于可以选择不同的连接位置（1→ 2, 1→ 3, 1→ 4, 1→ 6）、不同的异头构型（α 或 β）、不同的环大小（吡喃或呋喃）以及丰富的分支，竟然能组合出数万种截然不同的三维寡糖密码！

1. 糖蛋白（Glycoprotein）与两大糖肽键（年年必考填空与单选！）：
   糖蛋白（Glycoprotein）：由一个或几个较短但结构高度多样、常带分支的寡糖链通过共价键（N-糖苷键或 O-糖苷键）与蛋白质多肽链连接而成的糖缀合物；广泛分布于细胞膜表面和细胞外分泌液中（如免疫球蛋白 IgG、促卵泡激素 FSH、促甲状腺激素 TSH 等），参与蛋白质折叠质量控制、亚细胞定位、防蛋白酶降解及细胞间专一识别 2023·名词。（注：糖蛋白通常含糖量较少［1%～ 70%］，但也不可绝对化，例如红细胞膜上的血型糖蛋白 Glycophorin A 含糖量高达 60%）。
   寡糖链还原端异头碳与蛋白质氨基酸残基侧链之间的共价键称为糖肽键（Glycopeptide bond），严格分为两型：

3D3D 糖类：糖缀合物（蛋白聚糖、糖蛋白糖肽键与 ABO 血型）交互观察软骨蛋白聚糖瓶刷聚集体（糖 ≥95%）、糖蛋白 N-糖肽键(Asn)/O-糖肽键(Ser/Thr)、神经节苷脂与凝集素识别、ABO 血型寡糖抗原↗

• ① N-连接型糖肽键（N-linked）：由寡糖链还原端的 N-乙酰葡糖胺（GlcNAc） 以 β-构型通过 N-糖苷键 连接在多肽链特定共识序列 -Asn-X-Ser/Thr-（X 为除脯氨酸 Pro 外的任意氨基酸）中的 天冬酰胺（Asn） 侧链酰胺氮原子上 2017·填空！所有真核生物 N-连接寡糖链的还原端都共享一个完全相同的核心五糖（Man_3GlcNAc2）（在内质网中先由多萜醇焦磷酸载体 Dolichol-PP 组装十四糖前体，再整块转移给 Asn 并经高尔基体修剪加工为高甘露糖型、复杂型或杂合型）。
• ② O-连接型糖肽键（O-linked）：由寡糖链还原端的 N-乙酰半乳糖胺（GalNAc）（或半乳糖 Gal、甘露糖 Man 等）通过 O-糖苷键 连接在多肽链的 丝氨酸（Ser）或苏氨酸（Thr）（在胶原蛋白中还可连于羟赖氨酸 Hyl 或羟脯氨酸 Hyp）侧链的醇羟基氧原子上 2017·填空 2024·单选！（在高尔基体中逐个添加单糖合成，无共享核心五糖，无严格氨基酸共识序列，黏蛋白 Mucin 中极为丰富）。
• 考场必防陷阱：N-连接认准 天冬酰胺（Asn），千万别误选成谷氨酰胺（Gln）或赖氨酸（Lys）；O-连接认准带羟基的 丝氨酸（Ser）和苏氨酸（Thr）（酪氨酸 Tyr 虽然也带酚羟基，但它用于生糖原蛋白 Glycogenin 的自糖基化引物，常规糖蛋白 O-连接填空写 Ser、Thr）！

1. 糖脂（Glycolipids）、ABO 血型抗原决定簇与凝集素（Lectins）：
   除了连在蛋白质上，寡糖链还可以经糖苷键共价连在膜脂（神经酰胺 Ceramide 的 C1-OH）上形成鞘糖脂（Glycosphingolipids，如含唾液酸的神经节苷脂 Gangliosides）。无论是糖蛋白还是糖脂，其寡糖链在质膜上呈绝对不对称分布——100% 朝向细胞质膜的外表面（非胞质侧）！
   最经典的例子就是人类红细胞表面的 ABO 血型抗原决定簇：

3D3D 糖类：糖缀合物（蛋白聚糖、糖蛋白糖肽键与 ABO 血型）交互观察软骨蛋白聚糖瓶刷聚集体（糖 ≥95%）、糖蛋白 N-糖肽键(Asn)/O-糖肽键(Ser/Thr)、神经节苷脂与凝集素识别、ABO 血型寡糖抗原↗

• 决定人类 A、B、O 血型的化学本质不是蛋白质，而是红细胞膜鞘糖脂和糖蛋白末端的寡糖链决定簇！所有人（除极罕见的孟买血型外）都能合成基础的 H 抗原寡糖链（末端含 L-Fuc(α1→ 2)Gal-），这便是 O 型血的表面抗原；A 型血拥有 N-乙酰半乳糖胺转移酶，在 H 抗原的半乳糖上多加了一个 N-乙酰-D-半乳糖胺 GalNAc(α1→ 3)；B 型血拥有半乳糖转移酶，由于编码该酶的基因与 A 基因仅差几个氨基酸，它在同一位置多加了一个 D-半乳糖 Gal(α1→ 3)；AB 型血则同时表达两种转移酶，兼有 A、B 两种寡糖抗原！仅仅末端一个单糖的 C2 基团是 -NHCOCH₃（A 型）还是 -OH（B 型），就决定了输血时的生死相容性！
• 那么，细胞表面精妙的“糖密码”由谁来负责阅读？答案是 凝集素（Lectins）：一类广泛存在于动植物、微生物中，具有高度专一性糖结合结构域（能以非共价键高亲和力识别并结合特定寡糖结构）、但不具备酶催化活性也非抗体的蛋白质，是“阅读糖密码”、介导细胞间识别、黏附与信号转导的执行者（例如血管内皮细胞和白细胞表面的选择蛋白 Selectins 在炎症反应中识别白细胞表面唾液酸化寡糖，介导白细胞减速滚动并穿出血管奔赴感染灶；流感病毒血凝素识别宿主呼吸道上皮表面的唾液酸残基）。

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🎧 MIT 7.05 课堂原声英文实录对照（本节关联 1 讲 · 完整逐段实录）点击展开 / 收起 ▾

🎬 MIT 7.05 Lec 19 · Intro to Metabolism, Polysaccharides & Bioenergetics⏱ ~50:00

~00:00MITOCW | 19. Introduction Metabolism/Polysaccharides/Bioenergetics/Intro Pathways

~00:01MATTHEW

~00:02OK. So welcome back, everybody, to 705. I know this has been a really challenging semester for all of us. I want

~00:09VANDER

~00:09to particularly acknowledge for the seniors out there the tragedy of missing the end of your senior year.

~00:15HEIDEN:

~00:15Hopefully us, MIT can somehow make it up to you. I can say for my part, we will do our best to teach you biochemistry remotely. And just as a couple logistics, a reminder, I'm Matt Vander Heiden. I'm going to be covering the remaining part of 705. For the rest of the semester, we get to focus on biochemistry of metabolism. We're going to learn what metabolism is, why it's important. And basically, this can be quite a polarizing topic. In my experience, people tend to really love or really hate metabolism. It's interesting, because there are some people out there who pretend to hate it, but actually deep down really love it. Hopefully I will inspire you over the rest of the semester to at least have some appreciation for why metabolism is important. And regardless, for those of you who want to go to med school, this is a favorite topic of MCAT Exams. And also you will see that it will come up in many different areas of biology, regardless of whether you go to med school or not. So if you do anything further in biology, hopefully you'll find this material somewhat useful. OK, so getting into what metabolism is. So I think it's good to start with a textbook definition of metabolism. And so metabolism is the chemical reactions that all cells and organisms use to do two things. One, extract energy from the environment. And two, synthesize the macromolecules that make up all life. So really, what metabolism is then is the chemistry that makes life possible. And so understanding metabolism is really something that will help you better understand your food. There's lots of material out there in the popular press about what's healthy and what's not. At least this will allow you to form some of your own opinions about those claims, and how they relate to what really goes on in our cells and bodies. There's lots of implications for medicine, very relevant to agriculture, the energy sector, biofuels come from. And so metabolism really matters to tackle many of the challenges that face us in society. Now as a topic, this can be very daunting. As shown here on this image, we have hundreds, this is a typical metabolic pathway chart that is hanging in the walls of my office, many other laboratories and academic places around the world. If you look at this chart, it's filled with hundreds of enzymes, complex pathways. Our goal here is not to memorize this chart. You can always look up details of any pathway or reaction that you want. The goal here really is to see beyond this complexity. I want you to appreciate why metabolism works the way it does, why this chart is organized as it is. Because what we will find is that this chart is really variations on relatively few reactions. It basically is life repurposing similar chemistry to do many different things.

~02:53I think there is a beauty in how life can use these reactions to get such a diversity of macromolecules, and enable cells to solve all kinds of different problems. You will see that all of the pathways really follow common principles. These are shared across all species and all forms of life. And so at the chemical level, all life is really related. And this is why it's so relevant to understanding many of the challenges in medicine, understanding ecology, evolution. And for you MIT students, you'll see it's also very critical for using engineering approaches based on biology to solve various problems in the world. OK. So now let's start getting into this a bit more. Now, the first topic that we're going to cover over the next several lectures really relates to coming back to sugars and carbohydrates, which I talked about and introduced in my previous lecture. And we're going to do this because sugars and carbohydrates are key energy transduction molecules and cells. And it really facilitates a discussion of the principles for how metabolism works. Now our goal here isn't to memorize pathways. We could read about the glycolytic pathway in an hour. I could draw it up here in a very short period of time. Many of you have probably already done this in high school. Our goal is instead to understand this pathway at a deeper level, really see why the breakdown of sugars works the way it does, how cells use this to release energy, and how that can be used to support other cell functions. And so before we get to that, I want to start by introducing just a couple high-level concepts about metabolism. And so really, based on our definition before, metabolism is really about two things. It's about making stuff, and we have a fancy term for that. That is called anabolism. And it's about breaking stuff down. And the term for that is called catabolism. And anabolism, building stuff-- that is, producing biomass, growing all of the material that's out there, anabolic steroids help you grow. This is anabolism to build stuff. That is one side of metabolism. The other side is breaking stuff down, catabolism. That is, breaking down our food, eating food. Digesting our food is a way to get energy. Now of course, to reproduce, life has to build more cells. You need more stuff. You can't create something from nothing, and so that requires energy input. Anabolism typically requires energy input. Whereas that energy has to come from somewhere. And that's where catabolism comes from. We have to break down food. And ultimately, catabolism is the source for biological energy for many different systems. Now, some of this is actually somewhat intuitive. We all learned as little kids that we need to eat food if we're going to grow up and be big and strong. You also know that if you're going to run a race, you've got to eat a bunch of food, you need that energy to fuel your activity. But maybe what's less intuitive to many of you is we also need energy to sustain life, even if we're doing absolutely nothing. OK, adults, sit on the couch all day, hopefully they're not growing. But they'll still starve if they don't constantly eat some food, even if they're inactive. And so why is that the case? Well, hopefully some of you recall the second law of thermodynamics. So what is the second law of thermodynamics? It's, in effect, the entropy of the universe is increasing. That the universe continually tends towards disorder.

~06:05Life is exactly the opposite of this. Life is actually maintaining extreme order in the face of the second law. And so all life must constantly battle entropy. And many have described life at the very highest level as being really the ability to maintain order, to fight entropy, which of course requires constant energy input. And metabolism is the process that all cells use to do this. Now for humans, I think we all know we need to eat and breathe in order to stay alive. For you future MDs out there, what happens if your heart stops, or you stop breathing? Well, of course, you die in a matter of several minutes. Why is that? Well, that's because every cell in your body has to do constant catabolism in order to derive energy, in order to remain viable. This means they need constant food, and particularly oxygen delivery. And if you don't get those things, metabolism can no longer work, and the cells die. And so energy is sort of this mystical concept that we like to talk about. And so before we delve a little bit deeper into what I mean by biological energy, what it is, why we have to constantly do catabolism to maintain it, I want to go back and say just a little bit more about carbohydrates and finish our discussion there, and talk about carbohydrates and sugar polymers, polysaccharides, because this has some additional properties I didn't have time to cover in my prior lecture that is important to understand how it allows these molecules to store energy in a very efficient way. And it will also enable us to talk a little bit about how carbohydrates can also be used as structural molecules for lots of different organisms. All right, so a little diversion to discuss carbohydrates and polysaccharides. And so if you recall from my prior lecture, sugars that are greater than four to five carbons can exist either in open chain or ring forms, furanose or pyranose rings. I'll just remind you by redrawing up here, glucose. So this is D glucose, drawn in the open chain form. Remember this can form a ring. OK. So it's a reminder, carbon 1, 2, 3, 4, 5, 6. If we have this hydroxyl group from carbon 5, form a hemiacetal bond with the aldehyde at carbon 1, you get this ring structure. This would be alpha or beta, depending on whether the hydroxyl group here at carbon 1 is pointing up or pointing down. D-glucopyranose. So this is review of what we already talked about last time. Now, what I want to talk about is that if we form a disaccharide or a polysaccharide-- that is, begin to make sugar bonds, and if we do that in a way that ties up this hemiacetal or hemiketal bond, this then prevents the ring from opening and gaining access to that carbonyl, that aldehyde carbonyl, as this moves between the open chain and the ring form. And so a good example of this is the disaccharide sucrose. So sucrose is a disaccharide of glucose plus galactose. And here is what this disaccharide looks like. OK, so you have here, glucose in the alpha-D-pyranose form. Here's fructose in the beta, because the OH group is pointing out. Fructofuranose form. And so the formal name for this molecule, sucrose, would be alpha-D-glucopyranosyl 1-2, because we're going from carbon 1 of glucose to carbon 2, there's 1, 2, 3, 4, 5, 6 of fructose. Beta-D-fructofuranose. Sucrose or alphaD-glucopyranosyl 1-2 beta-D-fructofuranose.

~09:14OK, so this disaccharide forms a bond between carbon 1 and carbon 2. That would be the aldehyde in glucose, or the ketone tied up in the hemiacetal or the hemiketal bond of these two molecules. And so there's no way that either of these sugars can access the open chain form, without breaking this O-glycosidic bond. And that's one reason why sucrose is a very good storage molecule for carbohydrates, because without breaking that bond, you prevent access to gaining these reactive aldehyde or ketone bonds, that could react with some other molecule in the cell. Now, I mention this because whether or not a particular sugar is enabled to get access to this reactive aldehyde or ketone really forms the basis for a classic sugar detection lab test. And this is relevant to discuss this lab test because it explains some nomenclature that is still used, and in fact, we will continue to use a bit as we discuss some aspects of sugar metabolism. So what this test is, is basically, is that if you heat the sugar with copper, which is blue, and that copper can gain access to a free carbonyl--that is, if the sugar can access the open chain form to expose the aldehyde or a ketone, that copper can then become reduced to copper plus. That changes the color from blue to red. And so if you reduce the copper, you oxidize the bond there. And this test turns positive if there is a reducing sugar, is what it's called. So a sugar that reduces the copper from the blue 2 plus state to the red plus state is a positive test. And so basically, a reducing sugar is any sugar with an ability to access an open chain form. Because that will provide the free aldehyde or ketone to reduce the copper in this test. So this term, reducing sugar, obviously applies to all monosaccharides, because every single one of them has an aldehyde or a ketone, and can access the open chain form. But it will only apply to some disaccharides or polysaccharides. So let's come back to sucrose up there. So is sucrose a reducing sugar? No, it's not, because there is no way that you can access a free aldehyde or a ketone in sucrose, because it's tied up in that O-glycosidic bond between carbon 1 of glucose and carbon 2 of fructose. And so you need to break that bond to give them monosaccharide subunits in order to access the open chain form. However, let's give an example of a disaccharide that is a reducing sugar. And that's the disaccharide maltose. So maltose is basically two glucose molecules. And those two glucose molecules are like this. So this has a O-glycosidic bond between carbon 1 of this glucose, and carbon 4 of that glucose. And so formally, this would be alpha-D-glucopyranosyl 1-4 alpha or beta. If it's pointing down, it's alpha, pointing up, it's beta. Dglucopyranose. So maltose or alpha-D-glucopyranosyl 1-4 alpha or beta D-glucopyranose is a reducing sugar, because this sugar could access the open chain form. You could open up this carbonyl and expose the free aldehyde at position 1. Now, we could also refer to this sugar as having two different ends. We can refer to this end as being the nonreducing end, and this end as being the reducing end. Because of course, this bond, this aldehyde on the first glucose is tied up in this O-glycosidic bond, whereas the one on this one is not. So this end is non-reducing, and this end is reducing. Now this becomes much more relevant if we start talking about polymers. And so what starch is. So starch, what potatoes are made out of, is basically a polymer of maltose. Polymer of maltose molecules. So basically our glucose with 1-4 linkages.

~12:39OK, so I could draw that end, the reducing end of the polymer, in the alpha or beta. But this here, being the nonreducing end. Every other subunit is tied up in this 1-4 O-glycosidic bond. And so starch is really a polymer of glucose molecules with bonds between the 1 and the 4 position of each subunit. Now we will see later that we build and break down starch polymers from only one end, from the non-reducing end. And so having these terms, reducing and non-reducing, provides the term to specify different ends of a polymer. Reducing and non-reducing ends turns out to also be important for naming conventions for disaccharides or polysaccharides. And this becomes relevant if we consider the disaccharide lactose. So lactose is also a polymer of glucose plus galactose, except this polymer of glucose plus galactose, unlike sucrose, is different because it has a different linkage between the sucrose is a disaccharide of glucose plus fructose. Fructose, I wrote the name correctly. But lactose is a disaccharide between glucose and galactose. And so I'll draw it here. OK, so this is lactose. This is galactose here on the non-reducing end. So the formal name for this would be beta, because there's a beta linkage here between carbon 1 of this galactose and carbon 4 of this glucose. So be beta-D-galactopyranosyl 1-4. And this is glucose in the alpha or beta. D-glucopyranose. Lactose, or beta-Dgalactopyranosyl 1-4, alpha or beta, depending if I drew down, alpha, up, beta. D-glucopyranose. Now, this also has non-reducing and reducing ends. And by convention, you would name the sugar from the nonreducing end to the reducing end. Hence I put galactopyranosyl 1-4 alpha-D-glucopyranose, named them in that order. Now you'll note that lactose, unlike starch, unlike maltose, unlike sucrose, links these disaccharides with a beta linkage between this sugar and that sugar, where the other ones had alpha linkages. And structurally, this is very different. And I just want to point that out. So this here is basically alpha-D-glucopyranose. And so alpha, this is carbon 1. This is the hydroxyl pointing down. That's why it's alpha. If I make an O-glycosidic bond to another sugar, you can see that it points and creates this kinked structure. Now, if I were to make this beta, this hydroxyl rather than being here would be at this position on carbon 1. You can see that that now creates a very different geometry. Now you have a flat molecule, as opposed to a kinked molecule. Now, this has consequences, of course, for the enzymes that break down the sugars. Obviously, it's going to be a very different enzyme that breaks a bond in this orientation, versus a bond in that orientation. This has, basically implications for what enzymes we have. And so lactose, of course, is milk sugar. All mammals make milk, so make lactose, and break it down when we're babies. But most mammals tend to lose expression of the enzyme lactase that's able to break this 1-4 beta linkage as we age. And this is basically what accounts for lactose intolerance. Now, if you think about it, in the world, much of the world has issues with varying degrees of lactose intolerance in adulthood. The exception to this is often people of European ancestry. Why is this?

~15:36Well, Europeans historically drank milk well into adulthood. And so this selected for continuous lactase, the enzyme that wouldn't break that beta 1-4 linkage. The expression as people move into adulthood. And so it's an example of how cultural things, drinking milk into adulthood, really affected evolution, such that that subpopulation of people, people with that genetics to increase lactase expression, don't become lactose intolerant as they age. Now, this alpha versus beta linkage-- that is, whether or not you have this more kinked versus more flat structure-- also has a major effect on the structure of a polymer as well. And so if we look here at this starch polymer, it's basically this series of glucose molecules stuck together with alpha 1-4 linkages. So that's going to create a kinked structure. This is best shown here on this slide. So you can see this kinked structure. And so when you build this polymer, you're going to end up more with this helical structure in 3D space. That's a very efficient way to store glucose monomers in much less space than you would otherwise get with beta linkages. Now, it turns out that nature takes this a step further. And in addition to just having this starch polymer, it turns out sometimes you can add branches to the starch polymer to further increase the efficiency of energy storage. So if you have here a starch polymer, so let me just draw here a couple subunits. So this here is a starch polymer, glucose polymer with alpha 1-4 linkages that I can put an additional branch on this by adding on a linkage up here on the top. With an alpha 1-6 linkage. So the 1 position of here, to the 6 position of here, which has the effect of creating branches on this long polymer, such that you have a single reducing end, and lots of non-reducing ends. Each of these non-reducing ends has a polymer of alpha 1-4 linkages that would be stuck to the main chain polymer via this alpha 1-6 linkage. And this has a couple implications for storage. The first is, by only having one reducing end, it reduces maximally the exposure to a free aldehyde. And so that makes it a good storage sugar. And also as we will see, we're going to break down these sugar polymers biologically from the non-reducing ends, and build from those ends. And so there's lots of hooks or lots of places to either add or remove sugars from, which allows you to access carbohydrates quickly as needed. That's shown nicely here on this slide, better than I can draw it. So up here, you have your non-reducing end across the polymer. So this here would be starch with the alpha 1-4 linkages on the polymer, or reducing end on one side, non-reducing end on the other. You can then add branch points to that by making an O-glycosidic bond between carbon 1 down to carbon 6, creating a branch. And so you can see this helical branch structure will then form. That is both very efficient to pack lots of glucose into one place, and give lots of non-reducing ends with which to build and remove sugars from for biology to either store or quickly access sugar molecules as needed. Now, in plants, of course, potatoes use starch in a straight chain. We know that. However, plants also use this branched structure. This is a molecule called amylopectin. Amylopectin is better known in the food industry as Sure-Jell. It's the material that allows you to make jelly. It's the jelling agent in jelly. And effectively, this has every 24 to 30 units. There would be a branch with one of these alpha 1-6 linkages to give you this multi-branched structure.

~18:57Now, animals don't make starch. Animals don't make amylopectin. But animals make another molecule called glycogen. Glycogen, also a sugar polymer, exactly like amylopectin. Alpha 1-4 linkages with the alpha 1-6 to create branch points. Just like amylopectin, except glycogen has even more branches with a branch every 8 to 12 glucose units. And so, this is a very complex structure for both plants and animals to quickly store and quickly release glucose molecules when they're needed. Now, this is in contrast to a glucose polymer that can be done that has a very different 3D structure. So what if we take starch, and rather than have these alpha 1-4 linkages, but instead replace these with beta 1-4 linkages? OK, so now beta linkages. Looks simple enough, but changing the geometry from the alpha to the beta bond removes the kink. Now it's much more of a flat structure. Turns out this is, shown on an image here. So here's a beta linkage, and suddenly you have this flat structure. This is the same polymer as starch chemically, but has a different linkage, the beta 1-4 linkage. And that polymer with the beta 1-4 linkage is cellulose. And cellulose is, of course, what wood is made out of. And so wood and potatoes, cellulose and starch, exactly the same polymer. Same number of calories, if you could access the glucose units. But the alpha and beta bonds make them very different. Obviously, wood and cellulose is a very good structural polymer for plants. We build houses out of it. We don't build houses out of potatoes, but we eat a lot of potatoes. So equally good molecule for plants to store lots of food for energy. Turns out wood is a great source of energy too. You just need to have an enzyme that can break the beta 1-4 bond. Termites have symbiotic microbes that allow them to do this, and this is why termites can eat wood, a lot of energy tied up in wood. Now, we lack the time to go into the details about how this relates to other structural carbohydrates. But in general, structural carbohydrate or carbohydrate-like molecules form polymers of sugar or related molecules, also with these beta linkages. And a good example is chitin, so the material in the insect shells, is basically a sugar-like molecule that's a polymer with a beta linkage in it. Same thing with cartilage in humans and animals. And you can look up the details of what these things look like, of course, if you're interested. Chitin and cartilage aren't made of true polysaccharide polymers, but they're very related to polysaccharides. And they really illustrate how nature can take carbohydrate chemistry and repurpose it to basically act as an energy source, but also build all kinds of structural molecules that are really useful in biology. All right, now I want to go back to, here's ways that one can store and use carbohydrates in interesting ways. But now I want to come back to them as energy sources, and assuming you can access the glucose and whatever polymer it's present in, how can you actually metabolize it to provide energy in a way that sustains life? And this means we're going to turn to another topic, a topic that's typically referred to as bioenergetics. Bioenergetics, which is really the discussion of how energy is transduced in biological systems.

~21:58I'm going to introduce this topic today, we're going to revisit it throughout the course. But it's really important to ask this question. We really want to consider, what do we mean when we say biological energy? What is biological energy? Well, someone probably made you memorize in high school, and often you were thinking, oh, it's just ATP. And certainly, ATP, adenosine triphosphate, is a very important molecule for biological energy transduction. But I want to actually explore why ATP is actually useful. And if ATP is energy, why don't we just eat all kinds of ATP? I can say for a fact that absolutely none of you had ATP today for breakfast. No one sells ATP as an energy booster. And so if ATP is such a great energy source, why don't we just eat it? And to really understand ATP, how it works, what biological energy really is, we really need to revisit some very basic topics in thermodynamics.

~22:50Now, thermodynamics strikes fear into the hearts of students everywhere. You can get details of thermodynamics and the theory around these things in other courses. That's not really what we're going to try to accomplish here in 705. Our goal here is really a practical understanding of how thermodynamics applies to biology and metabolism. And we need to go into this, because it's key to understanding why we store energy, go through all this trouble to store energy as these carbohydrate polymers to begin with. Why we eat potatoes and not ATP, as well as why ATP is actually useful to cells. And so let's take a step back and just think about it. Let's think about wood. I just told you, wood is a polymer of glucose molecules with beta 1-4 linkages. How can we, forget as organisms, as cells. But how can you release energy from wood, to do other kinds of work? Well, we can burn it. And so what's the chemistry of burning wood? Well, it's a carbohydrate polymer. So it has this CnH2n formula. If we combine that carbohydrate with oxygen, it releases CO2 and water, plus some light, plus heat. This light and heat is energy, because we can use it to do work. Boil water, turn a turbine, make electricity. Whatever. Warm ourselves by the fire. Use the light to do some other things. And so this burning of wood is certainly release of energy. Now, we lack the enzymes to do this reaction in wood, because we can't break that beta 1-4 linkage. But we certainly can break that alpha 1-4 linkage in starch from potatoes. And we use the exact same chemistry to burn that glucose and release energy. Except the difference is that if I burn wood, I do this all in one step. But life is a much better engineer than that. It basically, rather than releasing all of that energy in one step, it really releases it stepwise in a manner that's actually useful to cells. But it's exactly the same release of energy. And so what life is, or extracting biological energy is, is it's really the ability to do stepwise oxidation of glucose or other carbon. I'll write glucose for now. To get energy. So we burn wood, that's favorable, it releases energy. If we do stepwise oxidation, we also can release energy as well. Same reaction, also releases energy.

~24:59How much energy do we get from releasing us burning glucose, as you do and burning it in a fire? Well, it has to be exactly the same amount, because it's exactly the same reaction. So we burn wood, we get light and heat. Cells burn glucose, it releases the exact same amount of energy. It just does so in a way that allows the cells to do biologically useful things. What are those? Well, it could be heat. All of us maintain temperature. So heat is a conversion of energy that of course, is useful to biology. But it can do other things, too. It can allow cells to move. It can allow them to do any reaction to fight that entropy that allows life to exist. And so all things, including ourselves, have to follow the same laws of thermodynamics. Life is not special in that regard. And so burning wood is favorable, because it increases the entropy of the universe. You're breaking up this polymer into a bunch of monomers. That's going to be spontaneous. That is in accordance with the second law of thermodynamics. True if cells burn it as well. But remember, the second law says that net entropy of the universe must always increase. And so if we're going to do something that's not energetically favorable, like build a glucose polymer, we obviously have to put energy into it. And to do so, the energy that's released must be greater than the energy that we actually store. And so this is actually a really key point, because anything that we do that requires energy input for a cell to carry out that process requires a source of energy release somewhere else that is equivalent or greater than what is actually put in. And so cells need energy, because they do lots of thermodynamically unfavorable things, fighting that entropy of the universe. And all of those processes must come from the release of energy elsewhere, like the burning of carbohydrate. And so that constant energy input is at the very highest level, why we need to do constant metabolism in order to maintain order and survive as organisms. Now this, of course, comes from things like burning of carbohydrate. But of course, that carbohydrate has to come from somewhere. And so ultimately, you have to have an external source. That external source is of course, the sun. Photosynthetic organisms can use light energy from the sun to do exactly the same things. Build those excess polymers, and that's why we as animals or anything that eats other animals as a way to live is ultimately depending on eating photosynthetic organisms, because photosynthetic organisms do this by harvesting energy of the sun. Now that's all very high level. But I want to come back to the specifics of metabolism, ultimately, and understand those as how they relate to enzymes' reactions and pathways. That is, how do you get that complex series of reactions that make life possible, and ultimately still be guided by these exact same principles? In other words, how do we couple the energy releasing reactions, like burning glucose, in a way that obeys the laws of thermodynamics and operates under biologically acceptable conditions in order to do things that are biologically unfavorable. That's really what bioenergetics is. And so the next thing we want to consider then, if we want to understand this, is really, let's ask the question. What determines if a specific reaction occurs? That is, what determines if something like burning wood actually spontaneously happens?

~28:08If you light wood on fire, it will burn. It does so every single time, everyone has had that experience. But no one has ever seen CO2 and water spontaneously come together and form a tree. That doesn't happen. So what determines whether you burn wood and that is favorable, versus why it is that CO2 and water don't come together and spontaneously form a tree? Do not think that this is determined entirely by enzymes. Enzymes are catalysts. Enzymes are the spark that makes the burning of wood possible. But remember, enzymes cannot change thermodynamics. Enzymes only change the rate at which a reaction happens. It does not change the equilibrium. That tree wants to, if given the catalyst, form CO2 and water. That reaction is spontaneous because it is thermodynamically favorable. That tree will not take CO2 and water and spontaneously reform a tree. And there's no amount of enzyme that will make that happen all by itself. And so remember that. If you have any reaction between A and B, there is some equilibrium, as defined by the chemical properties of A and B, such that the equilibrium lies far to one side or the other. And that equilibrium is determined by thermodynamics. It is not determined by enzymes. I don't care how much enzyme I add. If the equilibrium lies towards A as I draw it, I can add all the enzyme I want, and I can never create more B. And so, this is super important, and is actually a point that is gotten wrong by lots of biologists. They think, oh, this enzyme is expressed. Therefore, this pathway must be happening, or this reaction must be happening faster. You need an enzyme to catalyze a reaction. So a reaction may not occur, your wood may not burn unless you give it a spark, a catalyst to actually make it happen. But you cannot add a catalyst to fight thermodynamics. You cannot take that CO2 and water and turn it back into a tree. And the same thing is true. You can add all the enzyme in the world you want, and all it will do is help A and B establish the equilibrium that is defined by the thermodynamics of the relationship between A and B. Now, what determines the equilibrium between any two species? That's a topic for another class, why a species is favored to be one side or another. However, a useful tool to think about this in biochemistry and quantify this for any pair of reactions is something you've probably learned about before. It's something called the Gibbs free energy. Gibbs free energy, or delta G. Delta G is related to the way we'll talk about the equilibrium constant. And it's basically a term that we can use to specify if a reaction is favorable or not. And so hopefully you learned in an introductory class that if delta G is less than 0, our reaction is spontaneous. If delta G equals 0, our reaction is that equilibrium. If delta G is greater than 0, our reaction is not spontaneous. All right, if we take our log and ask, what is delta G to turn that log into CO2 plus water, it is less than 0, because it is very spontaneous. We take a pile of ashes and ask what delta G is to recreate the log. It's greater than 0, because it ain't going to happen. And if it's at some equilibrium, delta G is equal to 0. Now, delta G depends entirely on conditions, as we will see in a minute. And because of that, this means that there can be conditions where absolutely any reaction can be favorable if the conditions are right. And we'll see that this becomes very relevant to understand how metabolism works. And so let's go back and just now consider some generic reaction, A and B.

~31:37So at equilibrium, what happens? So at equilibrium, the concentration of A and the concentration of B are not changing. Delta G equals 0. These two things are at equilibrium, whatever that equilibrium is. A and B are not changing in concentration. Now if I come and I add more A to this side of the equation, what does that mean? Well, you know from Le Chatelier's principle that that's going to favor production of B. We have shifted things now out of equilibrium. So if I add A, that leads the production of B to re-establish this equilibrium. That means that delta G from A to B is less than 0 until I've reestablished equilibrium, and now delta G is back to 0 again. Now, what happens if I add B? Well, if I add B, now I'm going to produce some A. So add B. That leads the production of A, until I reestablish that equilibrium. So delta G, in that case, to go from B back to A. The reverse reaction is less than 0. Or I could also say that delta G, to go to A to B here, because that's not going to happen. I just added more B. I'm not going to suddenly create more B from A. So here now, delta G is greater than 0. Now note when I did this, I don't actually have to specify how much A or how much B I've added. The equilibrium depends on the ratio of B to A. Not the absolute concentration of either species. And so having a way to think about this equilibrium, it turns out, is also useful for biological systems to divine some delta G that is helpful to relate to this equilibrium constant. And that is this concept of delta G 0 prime. A standard free energy, which for biological systems, relates to the equilibrium constant at 25 degrees, or the pH of 7 and 1 atmosphere. Pretty typical biological conditions. And this here is basically related to the equilibrium constant. And we can calculate the actual delta G by the following formula. Delta G equals delta G 0 prime plus RT times the log of the products over the reactant. So this would be drawn for the reaction A to B. So the reaction A to B is related to delta G 0 prime, which is related to the equilibrium constant. I'll tell you how in the second. Plus R is the gas constant, T is the temperature in Kelvin times the natural log of the ratio of products, B over reactants, A. This tells you whether or not at specific concentrations of B and A, that is a specific ratio of B and A, whether it is favorable to go from A to B, delta G less than 0, or whether it is favorable to go from B to A, delta G greater than 0. And so this should begin to make it clear that whether or not a specific reaction occurs is affected by the actual conditions present. And you can calculate that based on this relationship. And so if I specify concentrations of A and B, as well as delta G 0 prime, I can know, at those concentrations, which direction of the reaction is favored. Now of course, my drawing A to B is entirely arbitrary. I could equally draw it B to A. And if I did that, so this is delta G 0 prime, A to B, all I do is flip the signs. So delta G equals delta G 0 prime. The negative of A to B is equal to the positive of delta G 0 prime from B to A, because the direction is arbitrary. Plus RT log in this case. Now I have A is my product, and B is my reactant. If I just flip the sign, flip that ratio, it's just going to change the sign of my product and give me exactly the same result with the opposite sign, which makes sense. What's favorable in one direction is not favorable in the other direction, and vice versa.

~35:17Now, I can also set delta G equals 0. That's equilibrium. If I do that, then delta G 0 prime equals negative RT. So this would be for A to B. I'll use the top example. So delta G equals 0. Then I have delta G 0 prime equals RT times log B over A. So if I know two things are at equilibrium, I can calculate delta G 0 prime, and know what the equilibrium constant is. And so it follows from this then that if delta G 0 prime is negative, that means B is favored over A at equilibrium. And if delta G 0 prime is positive, that means A is favored over B at equilibrium.

~35:55And so delta G 0 prime really is a convenient way to look at a reaction, and know which direction the equilibrium lies. So A and B, delta G 0 prime is negative, equilibrium lies to B. Between A and B, if delta G 0 prime is positive, the equilibrium lies towards A. But the key concept is that for any specific reaction, any specific conditions of A and B, whether or not A is converted to B will be defined by the equilibrium constant. But it will also be defined about what delta is under those conditions. So to be clear, delta G will depend on conditions, because I'll write it again, delta G equals delta G 0 prime plus RT times the log for the reaction, A goes to B. So products over reactants. And so whether or not A is converted to B will be a property of the equilibrium constant, plus the concentration of B and the concentration of A, that ratio of concentrations at the conditions present. And if in that calculation, delta G is less than 0, that reaction is spontaneous. If delta G is greater than 0, that reaction is not spontaneous. Or to put it another way, if delta G is less than 0 under those conditions, energy is released. Or if delta G is greater than 0, that reaction will not occur without energy input. If I burn wood, energy is released. But there's no way I can reassemble that log without some kind of energy input. And really getting this concept is central to understanding metabolism, as well as what biological energy means. And hopefully, what is now apparent, based on what I just said, is that what really determines delta G is two things. Of course it's the equilibrium constant. But in essence, delta G for any reaction is proportional to the ratio of the reactants over the products. And that I can come up, at least in theory, with any relationship, any ratio of reactants to products to make this favorable. At some tiny, tiny concentration, if I have nothing but CO2 and water, and infinite time and infinite catalysis, a tiny bit of wood could spontaneously form. And so life really creates the conditions that allow that, and select for that to happen, and of course, add energy input so it can actually happen more. And so this should be very clear to you that absolutely no reaction is irreversible. I don't care what your textbook says. Lots of sources will discuss irreversible reactions. I'll talk about irreversible reactions later in the course. But what we mean when we say the word irreversible reaction is these are under conditions that are found in cells and in nature. And I stress this because if we want to understand the energetics for how pathways work, we have to appreciate that life can create conditions to do unfavorable things, because that's the only way that pathways can work.

~38:33All right, what what do I mean by that? Well, suppose again, we'll come back to our reaction. A goes to B. Suppose I need to build a pathway where I need to convert A to B, because B is actually useful for some purpose that I need to do in a cell. However, what if this is at least by equilibrium, unfavorable? What if the equilibrium lies towards A? That means delta G 0 prime is positive, is greater than 0, for A to B. What does that mean? Delta G 0 prime greater than 0, for the reaction A to B? That means the equilibrium lies towards A. And so there is no amount of enzyme I can add that will allow me to net convert A to B, because the equilibrium lies far towards A. However, whether or not a cell is able to build a pathway, where it converts A to B depends on delta G, not the equilibrium constant for that specific reaction. So how can I turn A into B? Well, I could turn A into B if I keep the concentration of B low enough that delta G, not delta G 0 prime, that's the equilibrium constant, but delta G favors A to B conversion? That is, delta G for A to B is less than 0. How can I do that? Well, remember delta G equals delta G 0 prime plus RT log of B over A. So this term is positive, because I told you, the equilibrium lies towards A. So I need a concentration, a ratio of B to A, such that B is low enough that this term is more negative than that term is positive. And if that's the case, delta G will be less than 0, and I can net move that forward. So how can I do that? How can I keep the concentration of B low? Well, I can build a pathway that consumes B in a way to keep it low, which favors A to B conversion. In other words, I can build a pathway, A goes to B goes to C. So even here, where delta G 0 prime is greater than 0, if delta G 0 prime for this reaction, B to A, is much less than 0, such that equilibriums strongly favor C, that means my unfavorable equilibrium here can be overcome by the very favorable equilibrium of B to C. In essence, I can use B to C to pull A to B, such that it actually happens. Produce this intermediate that's useful on the path to making C. This strategy is used in lots of metabolic pathways. And it's useful to generate lots of chemically useful intermediates. Now note that this only will work if conversion of A to C is favorable. That is, if delta G 0 prime is less than 0. I can only pull this reaction if delta G 0 prime is less than 0. That is, if equilibrium favors A to C, I can build a pathway this way to do something unfavorable on the way to making C. This brings up another key point, in that everything that we discuss, relationship between any two metabolites in any single reaction, also must be true for entire pathways. So I have a three-step pathway from A to C. Whether I turn A into C in one step, or turn A into C in multiple steps, the free energy is exactly the same. If I burn wood by lighting it on fire, it releases the same amount of energy than if a termite has an enzyme that breaks that beta 1-4 linkage in the wood, and can burn that glucose stepwise through metabolism, the exact same amount of energy is released. Glucose on one side, CO2 and water on the other side, exactly the same amount of energy released, whether I do it in one step, or I do it in multiple steps. The equilibrium constant between those has to be the same, and the delta G has to be the same between all of it.

~42:13And so it should become apparent, then, that A to C has to be favorable if I'm going to use this trick of keeping product low in order to pull an otherwise unfavorable reaction forward. In other words, there's no way for me to keep B low and net convert A to B all on its own. That will not work. And it's these unfavorable reactions that ultimately then require energy input. And this is where ATP now starts becoming useful for cells. OK, so now I want to talk a little bit about how ATP provides energy that allows otherwise unfavorable reactions to occur. And ATP is useful because delta G applies to sets of reactions in the same way it applies to single reactions. That is, it applies to whole systems. All reactions are a series of reactions, whether they happen alone or coupled together. All have to follow the same rules. So now let's make our reaction more complicated. Let's say A plus B goes to C plus D, where this is really two reactions coupled together. A being turned into C, and B being turned into D. All right, so will this reaction happen? Can I turn A plus B into C plus D? Well, how do I know? Well, it's going to be, got to calculate delta G. And so it's going to be delta G 0 prime for A going to C, plus delta G 0 prime for B going to D. So those will relate to what's the equilibrium constant, the equilibrium relationship between A and C and B and D. If it's less than 0, equilibrium will favor to the right. If it's greater than 0, equilibrium will favor to the left of A and C and B and D. Add them together, that gives me my overall delta G 0 prime for those two reactions. Plus RT times the log of the products. So that's C and D over A and B. Products over the reactants. So if I take an unfavorable reaction-- say, A to C, if delta G 0 prime is greater than 0, so it would favor A, and I couple it to another reaction, B to D that's very favorable, because I add these two terms together. I can now take something where the equilibrium would be unfavorable, and make it favorable. However, whether or not this actually happens still depends on this ratio of products to reactants. So it still depends on the ratio of C to A, and D to B in our hypothetical thing here. So to know if a specific reaction will actually occur, we have to take into account both the sum of the delta G primes related to the equilibrium constant, and the actual ratios that are present in cells. And so the net effect is that I can couple more favorable reactions to make otherwise unfavorable reactions now become favorable. That is, have energy input. But it still depends on conditions. And so let's think a little bit about the polymer synthesis that we learned from Professor Yaffe. And so in that process, what did we do? We made several polymers, and you learned about the chemistry to do this. We made DNA, we made RNA, and we made protein. These are polymers of nucleic acids. Protein is a polymer of amino acids. All of these fight entropy, right? We're building polymers, not breaking them down. And all of them were synthesized using reactions that hydrolyzed ATP. In fact, those reactions actually hydrolyzed ATP in the following way. And every single one of those cases, if you look back, what you will see is that they all had steps where ATP was taken to AMP plus 2 inorganic phosphates. This reaction is very favorable. In fact, it's two reactions. It's really ATP goes to AMP plus pyrophosphate.

~45:39And then that pyrophosphate goes to 2 inorganic phosphates. It turns out that delta G 0 prime is less than 0 for both of these reactions. That means the equilibrium lies towards AMP plus pyrophosphate. And here, the equilibrium lies towards 2 inorganic phosphates. And so there's actually two tricks here by metabolism. One is, it's two very favorable reactions that we're coupled to do something unfavorable, build a polymer. And use the trick of keeping pyrophosphate low by coupling it to a downstream, very favorable reaction, which even further pulls this reaction, and is why ATP hydrolysis here becomes so useful as a way to build these polymers. Let's just go quickly and add a few numbers to show exactly what I mean by how ATP hydrolysis can be useful in this setting. And so, let's do something a little simpler. Let's go to ATP plus ADP plus to inorganic phosphate. So let's give this some numbers, delta G 0 prime in this case is equal to negative 7.5 kcals per mole. All right, what does this mean? Delta G 0 prime is related to the equilibrium constant. It's less than 0, which means the equilibrium lies towards ADP plus phosphate. Now let's see how this actually helps by considering what the first step in glucose metabolism by cells is. So the first step cells do in glucose metabolism is add an inorganic phosphate group to glucose. So it's this reaction, glucose plus inorganic phosphate goes to glucose phosphate. Just a quick note on shorthand that I will use in this course. So PI, of course, is inorganic phosphate. That is this PO43 minus group. When it forms a phosphodiester bond, like to an alcohol and one of the glucose molecules, I'll draw this in a future lecture. I'll oftentimes indicate it by putting a circle around the phosphate. And so adding this phosphate to glucose is a step that traps that glucose in cells. For now, let's just consider this reaction. And so delta G 0 prime of this reaction is positive 3.3 kcals per mole. What does that mean? Delta G 0 prime relates to the equilibrium constant. It says the equilibrium constant lies here to the left, to the glucose plus phosphate side. So if we want to net trap glucose phosphate in cells, well, it's not going to happen spontaneously. You need some kind of energy input. That can come from ATP. And so if we couple those two reactions as follows. So we now have glucose plus ATP goes to glucose phosphate plus ADP. Now let's draw this out. Will this happen? So we can calculate delta G. Delta G equals delta G 0 prime. So there's two reactions happening here, glucose to glucose phosphate, and ATP to ADP. We know what delta G 0 prime is by adding those together. So it's 3.3 plus negative 7.5. That equals negative 4.2. What does that tell us? That says, now we've created conditions where the equilibrium constant lies towards the right, towards the glucose phosphate plus ADP side. But how much of this occurs or whether it occurs isn't just the property the equilibrium constant. It's also how much is there. And so that's RT times the log of the products, glucose phosphate.

~48:34And ADP over glucose. And ATP. And so, if this term is less than 4.2-- that is, if the ratio of glucose phosphate to glucose and ADP to ATP in this term ends up being less than positive 4.2, this reaction is spontaneous. It is favored. Lots of conditions where that would be the case, although if this term is 4.2 or greater, now it will no longer occur. How much ATP is needed? Well, it's hard to answer this question in absolute terms, because if there's a lot of ADP around, you need a lot more ATP for the ADP to be useful. And this is the problem with equating ATP with energy. The energy here is in the ratio of ATP to ADP, or ADP to ATP. And this is something that we we'll come back to in the next lecture, because in the end, this is what will determine if a reaction has happened. It is not the absolute concentration of ATP. It's the fact that ATP hydrolysis is favorable. But how favorable that is depends on the relevant concentrations of ATP or ADP. And relative concentrations are all that matters in thermodynamics. It is not absolute concentrations. It is the ATP-ADP ratio that is the correct way to think about energy. And this is true for any reaction with respect to energetics. If an ATP-ADP ratio exists, such that when coupled to a reaction, delta G is less than 0, then it becomes spontaneous. And this is how ATP provides energy. And we will come back to this more in the next lecture. Thank you.

1.5新陈代谢总论与生物能学热力学定律：为什么酶救不了逆热力学的反应，而生命却能逆流而上？

一、新陈代谢（Metabolism）的定义、基本要略、两用途径与研究方法

当我们在前四节理清了糖类的化学结构与多糖储库后，Vander Heiden 教授在 L19 课堂中央挂起了一幅密密麻麻、包含数百条反应与酶的罗氏生化代谢全图（Metabolic pathway chart），微笑着对全班说：“很多人看到这张图的第一反应是恐惧，以为生物化学就是把这几百个化学式背下来。但我们这门课的目标恰恰相反——我们要看穿这张复杂网络的底层逻辑！你会发现，整张代谢图不过是少数几种有机化学反应（氧化还原、基团转移、羟醛缩合、异构化、水解）的重复变奏，而且每一条途径都严格服从完全相同的热力学法则。”

在王镜岩《生物化学》第 4 版与华农考研大纲中，首先要求你一字不差地掌握新陈代谢的核心框架：

1. 新陈代谢的标准定义：
   新陈代谢（Metabolism）：泛指生物体与外界环境之间的物质和能量交换，以及生物体自身内部的物质和能量转变过程，是生命现象的最基本特征。
2. 分解代谢（Catabolism）与合成代谢（Anabolism）的对立统一：

• 分解代谢（Catabolism，降解放能）：将复杂的营养大分子（糖、脂质、蛋白质）逐步氧化分解为简单小分子终产物（CO₂、H_2O、NH₃、尿素），是一个氧化、放能（Exergonic）的过程，释放的自由能被捕获用于合成 ATP 及还原力 NADH / FADH₂ / NADPH。
• 合成代谢（Anabolism，生物合成耗能）：利用小分子前体（构造元件 Building blocks）合成核酸、蛋白质、多糖、脂质等生物大分子以组装细胞结构，是一个还原、耗能（Endergonic）的过程，必须依赖分解代谢提供的 ATP 和还原力 NADPH 驱动。
• 新陈代谢的三大基本要略（填空高频考点）：新陈代谢的基本要略是：形成 ATP（通用能量通货）、还原力（主要为 NADPH） 和 小分子构造元件（碳骨架前体），用于生物合成与维持生命活动！

1. 两用代谢途径（Amphibolic pathway）：
   并非所有途径都能非黑即白地划入纯分解或纯合成。两用代谢途径（Amphibolic pathway）：在细胞代谢网络中，既参与分解代谢以氧化产能，又为合成代谢提供小分子前体碳骨架的中心代谢途径，最典型的代表是三羧酸循环（TCA 循环 / 柠檬酸循环） ⚑华农高频（例如 TCA 循环既能将乙酰-CoA 彻底氧化为 CO₂ 产生 ATP，其草酰乙酸、α-酮戊二酸、琥珀酰-CoA 又能随时被抽走去合成天冬氨酸、谷氨酸、葡萄糖和血红素卟啉）。
2. 新陈代谢的三级水平调节与研究方法：

• 三级调节体系：① 分子水平调节（最基础，包括酶活性的快速调节［别构调节、可逆共价修饰如磷酸化/去磷酸化、酶原激活、聚合解聚］和酶含量的缓慢调节［基因转录诱导与阻遏、蛋白质降解］）；② 细胞水平调节（真核生物细胞器区室化 Compartmentation 与跨膜转运控制，如脂肪酸 β-氧化在线粒体而合成在胞质）；③ 整体水平调节（高等动物通过激素［胰岛素、胰高血糖素、肾上腺素］和神经系统协调各器官代谢分工）。
• 研究新陈代谢途径的六大实验方法 2022·简答：① 使用专一酶抑制剂阻断某步反应，测定上游积累的中间产物（如用碘乙酸阻断 GAPDH 发现果糖-1,6-二磷酸）；② 利用遗传代谢缺陷症或营养缺陷型突变株研究（如 Garrod 研究尿黑酸症）；③ 同位素示踪法（如用 ¹⁴C、³H、¹⁵N 追踪碳氮原子去向，卡尔文用 ¹⁴CO₂ 揭示光合碳循环）；④ 核磁共振波谱法（NMR）（无创实时追踪活体代谢物）；⑤ 色谱-质谱联用技术（GC-MS / LC-MS）与代谢组学；⑥ 基因敲除（Knockout）、RNA 干扰（RNAi）与转基因技术。
• 代谢组学（Metabolomics）：在基因组学和蛋白质组学之后发展起来，系统研究生物体、组织或细胞内全部小分子代谢物（代谢组）的组成、动态变化规律及其与生理病理功能关系的学科，常采用核磁共振（NMR）及色谱-质谱联用（GC-MS / LC-MS）进行高通量定性定量分析 2019/2022·名词。

二、热力学第一性原理推演：为什么人躺在沙发上一动不动也会饿死？

在 L19 课堂上，Vander Heiden 教授提出了一个看似调侃却极为深刻的问题：“我们都知道跑步需要能量，长身体需要能量。但如果一个成年人整天躺在沙发上一动不动、体重也不再增长，为什么他不吃饭还是会饿死？如果心脏停跳或停止呼吸，为什么几分钟内脑细胞就会全部死亡？”

答案藏在热力学第二定律（The Second Law of Thermodynamics）里：宇宙的总熵（Entropy，无序度 S）总是自发地趋向增加。而生命（Life）恰恰是在宇宙熵增的洪流中强行维持高度有序状态的逆行者（维持跨膜离子梯度、不断修复受损 DNA、更新降解错折蛋白质）。根据热力学第二定律，要想在细胞局部维持高度有序（减熵），就必须每时每刻从外界摄入富含自由能的营养物质，将其氧化分解并向环境释放热量和无序小分子（CO₂ 和 H_2O），使整个宇宙的总熵增加！一旦营养氧化或氧气供应中断，细胞对抗熵增的自由能输入瞬间归零，无序度立即吞噬细胞，这就是死亡的热力学本质。

为了定量判断任何一个生化反应（或整条代谢途径）能否自发进行，美国物理化学家 Gibbs 引入了吉布斯自由能变（Gibbs free energy change，Δ G）：

Δ G = Δ H - TΔ S

其中 Δ H 为焓变（反映化学键断裂与形成的热量变化），T 为绝对温度（K），Δ S 为熵变。在恒温恒压下：

• 当 Δ G < 0 时：反应为放能反应（Exergonic reaction），在热力学上可以自发正向进行；
• 当 Δ G = 0 时：反应处于化学平衡状态（Equilibrium），正逆反应速率相等，反应物与产物净浓度不再改变；
• 当 Δ G > 0 时：反应为吸能反应（Endergonic reaction），在热力学上不能自发正向进行（其逆反应自发），但在酶的催化下，与之相偶联的强放能反应可驱动这类反应进行 2012/2018·判断。
• 命题核心铁律：酶（Enzyme）作为生物催化剂，只能通过降低反应活化能（Δ G^‡）来加快反应达到平衡的速率，绝对不能改变反应的吉布斯自由能变（Δ G）和化学平衡常数（K'_eq），更不能使热力学上 Δ G > 0 的反应单独自动发生！

更重要的一条热力学铁律是：吉布斯自由能 G 是状态函数（State function），Δ G 只取决于反应的始态（反应物）和终态（产物），而与反应经历的具体路径或步数完全无关（盖斯定律 Hess's Law）！

Vander Heiden 教授在黑板上写下了燃烧葡萄糖的总反应式：

C_6H12O₆ + 6 O₂ ⟶ 6 CO₂ + 6 H_2O Δ G'^° = -2870 kJ/mol（即 -686 kcal/mol）

无论你是把一块木头或一勺葡萄糖扔进火炉里一步烧光（2870 kJ/mol 自由能瞬间全部变成刺眼的光和滚烫的热），还是在细胞内通过糖酵解、丙酮酸脱氢、TCA 循环和呼吸链这几十步酶促反应逐步氧化（Stepwise oxidation），只要起点都是葡萄糖 +O₂、终点都是 CO₂ + H_2O，它们释放的总自由能分毫不差，全都是 -2870 kJ/mol（-686 kcal/mol）！生命的工程学天才之处，就在于把这 -2870 kJ/mol 的巨大能量瀑布拆解成几十级温和的小台阶，从而在恒温（37^°C）下将能量一节一节地捕获到高能化学键中，而不把细胞烧成灰烬！

三、标准自由能变 Δ G'^° vs 实际自由能变 Δ G：生命逆流而上的两大热力学法宝

在生化和考研计算中，最核心也最容易出错的公式，就是生化标准自由能变（Δ G'^°）与细胞内实际自由能变（Δ G）之间的关系！让我们跟着 Vander Heiden 教授在 L19 和 L20 的黑板板书一步步推导：

1. 什么是生化标准状态（Δ G'^°）？
   在普通化学标准状态（Δ G^°）中，所有溶质（包括 H⁺）的标准浓度均规定为 1 mol/L，这意味着 pH = 0（强酸环境），活细胞早就被强酸腐蚀殆尽了！因此，生物化学家专门定义了生物化学标准状态（Biochemical standard state）：

• 生化标准状态规定：温度 T = 298.15 K（25^°C），气压 1 atm（101.3 kPa），pH = 7.0（即 [H⁺] = 10^-7 mol/L 恒定），水（H_2O）的活度记为 1（默认 55.5 mol/L 已并入常数），除 H⁺ 和水以外的所有反应物和产物的初始浓度均为 1 mol/L（若反应需要 Mg²⁺，通常规定 [Mg²⁺] = 1 mmol/L）。在此标准状态下测得的自由能变记作 Δ G'^°（平衡常数记作 K'_eq）。

1. 实际自由能变方程与平衡常数关系：
   对于任意生化反应 A + B ⇌ C + D，在细胞内的非标准浓度下，其实际自由能变 Δ G 由下式决定：
   Δ G = Δ G'^° + RTln([C][D])/([A][B])
   其中 R = 8.314 J/(mol·K)（或 1.987 cal/(mol·K)）为摩尔气体常数，T 为绝对温度。
   当反应达到平衡状态（Equilibrium）时，净反应停止，此时 Δ G = 0，且产物与反应物的平衡浓度比值恰好等于生化平衡常数 K'_eq = ([C]_eq[D]_eq)/([A]_eq[B]_eq)。代入上式立即得到：
   0 = Δ G'^° + RTln K'_eq ⇒ Δ G'^° = -RTln K'_eq = -2.303 RTlg K'_eq

既然细胞内有大量生化反应（如合成大分子、糖酵解中的醛缩酶裂解、TCA 循环中的苹果酸脱氢酶反应）的 Δ G'^° > 0（即标准平衡偏向反应物一侧），细胞在不能篡改 Δ G'^° 的前提下，究竟用什么魔法让实际 Δ G < 0，从而逼着反应正向进行？Vander Heiden 教授在黑板上总结了生命逆流而上的两大热力学法宝：

让我们完整复现 Vander Heiden 教授在 L19 结尾与 L20 开篇黑板上推导的经典偶联算账案例——糖酵解第一步：把葡萄糖锁在细胞内（葡萄糖磷酸化）：

• 半反应 ①（吸能，不利）：要把无机磷酸加到葡萄糖 C6 羟基上合成 6-磷酸葡萄糖（G-6-P），属于酯化缩合：
  葡萄糖（Glc） + P_i ⟶ 6-磷酸葡萄糖（G-6-P） + H_2O Δ G'^°₁ = +13.8 kJ/mol（课堂板书 +3.3 kcal/mol）
  因为 Δ G'^°₁ > 0，平衡远远偏向左边的游离葡萄糖，单靠葡萄糖和 P_i 绝不可能自发生成 G-6-P。
• 半反应 ②（放能，极有利）：ATP 水解脱去末端磷酸：
  ATP + H_2O ⟶ ADP + P_i Δ G'^°₂ = -30.5 kJ/mol（课堂板书 -7.5 kcal/mol）
• 己糖激酶（Hexokinase）催化的偶联总反应（① + ②，消去 P_i 和 H_2O）：
  Glc + ATP ──(己糖激酶, Mg²⁺)──► G-6-P + ADP
  Δ G'^°_总 = Δ G'^°₁ + Δ G'^°₂ = +13.8 + (-30.5) = -16.7 kJ/mol（课堂板书 +3.3 - 7.5 = -4.2 kcal/mol）
  看！通过将磷酰基直接从 ATP 转移给葡萄糖，总反应的 Δ G'^°_总 变成了显著的负值（-16.7 kJ/mol），标准平衡常数 K'_eq = e^{-Δ G'°/RT} ≈ 850！再结合细胞内维持的高 [ATP]/[ADP] 比值，其实际 Δ G = -16.7 + RTln([G-6-P][ADP])/([Glc][ATP]) 高达 -33 kJ/mol 左右，在生理条件下成为一步彻底单向、不可逆的“捕糖陷阱”！

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🎧 MIT 7.05 课堂原声英文实录对照（本节关联 2 讲 · 完整逐段实录）点击展开 / 收起 ▾

🎬 MIT 7.05 Lec 19 · Intro to Metabolism, Polysaccharides & Bioenergetics⏱ ~50:00

~00:00MITOCW | 19. Introduction Metabolism/Polysaccharides/Bioenergetics/Intro Pathways

~00:01MATTHEW

~00:02OK. So welcome back, everybody, to 705. I know this has been a really challenging semester for all of us. I want

~00:09VANDER

~00:09to particularly acknowledge for the seniors out there the tragedy of missing the end of your senior year.

~00:15HEIDEN:

~00:15Hopefully us, MIT can somehow make it up to you. I can say for my part, we will do our best to teach you biochemistry remotely. And just as a couple logistics, a reminder, I'm Matt Vander Heiden. I'm going to be covering the remaining part of 705. For the rest of the semester, we get to focus on biochemistry of metabolism. We're going to learn what metabolism is, why it's important. And basically, this can be quite a polarizing topic. In my experience, people tend to really love or really hate metabolism. It's interesting, because there are some people out there who pretend to hate it, but actually deep down really love it. Hopefully I will inspire you over the rest of the semester to at least have some appreciation for why metabolism is important. And regardless, for those of you who want to go to med school, this is a favorite topic of MCAT Exams. And also you will see that it will come up in many different areas of biology, regardless of whether you go to med school or not. So if you do anything further in biology, hopefully you'll find this material somewhat useful. OK, so getting into what metabolism is. So I think it's good to start with a textbook definition of metabolism. And so metabolism is the chemical reactions that all cells and organisms use to do two things. One, extract energy from the environment. And two, synthesize the macromolecules that make up all life. So really, what metabolism is then is the chemistry that makes life possible. And so understanding metabolism is really something that will help you better understand your food. There's lots of material out there in the popular press about what's healthy and what's not. At least this will allow you to form some of your own opinions about those claims, and how they relate to what really goes on in our cells and bodies. There's lots of implications for medicine, very relevant to agriculture, the energy sector, biofuels come from. And so metabolism really matters to tackle many of the challenges that face us in society. Now as a topic, this can be very daunting. As shown here on this image, we have hundreds, this is a typical metabolic pathway chart that is hanging in the walls of my office, many other laboratories and academic places around the world. If you look at this chart, it's filled with hundreds of enzymes, complex pathways. Our goal here is not to memorize this chart. You can always look up details of any pathway or reaction that you want. The goal here really is to see beyond this complexity. I want you to appreciate why metabolism works the way it does, why this chart is organized as it is. Because what we will find is that this chart is really variations on relatively few reactions. It basically is life repurposing similar chemistry to do many different things.

~02:53I think there is a beauty in how life can use these reactions to get such a diversity of macromolecules, and enable cells to solve all kinds of different problems. You will see that all of the pathways really follow common principles. These are shared across all species and all forms of life. And so at the chemical level, all life is really related. And this is why it's so relevant to understanding many of the challenges in medicine, understanding ecology, evolution. And for you MIT students, you'll see it's also very critical for using engineering approaches based on biology to solve various problems in the world. OK. So now let's start getting into this a bit more. Now, the first topic that we're going to cover over the next several lectures really relates to coming back to sugars and carbohydrates, which I talked about and introduced in my previous lecture. And we're going to do this because sugars and carbohydrates are key energy transduction molecules and cells. And it really facilitates a discussion of the principles for how metabolism works. Now our goal here isn't to memorize pathways. We could read about the glycolytic pathway in an hour. I could draw it up here in a very short period of time. Many of you have probably already done this in high school. Our goal is instead to understand this pathway at a deeper level, really see why the breakdown of sugars works the way it does, how cells use this to release energy, and how that can be used to support other cell functions. And so before we get to that, I want to start by introducing just a couple high-level concepts about metabolism. And so really, based on our definition before, metabolism is really about two things. It's about making stuff, and we have a fancy term for that. That is called anabolism. And it's about breaking stuff down. And the term for that is called catabolism. And anabolism, building stuff-- that is, producing biomass, growing all of the material that's out there, anabolic steroids help you grow. This is anabolism to build stuff. That is one side of metabolism. The other side is breaking stuff down, catabolism. That is, breaking down our food, eating food. Digesting our food is a way to get energy. Now of course, to reproduce, life has to build more cells. You need more stuff. You can't create something from nothing, and so that requires energy input. Anabolism typically requires energy input. Whereas that energy has to come from somewhere. And that's where catabolism comes from. We have to break down food. And ultimately, catabolism is the source for biological energy for many different systems. Now, some of this is actually somewhat intuitive. We all learned as little kids that we need to eat food if we're going to grow up and be big and strong. You also know that if you're going to run a race, you've got to eat a bunch of food, you need that energy to fuel your activity. But maybe what's less intuitive to many of you is we also need energy to sustain life, even if we're doing absolutely nothing. OK, adults, sit on the couch all day, hopefully they're not growing. But they'll still starve if they don't constantly eat some food, even if they're inactive. And so why is that the case? Well, hopefully some of you recall the second law of thermodynamics. So what is the second law of thermodynamics? It's, in effect, the entropy of the universe is increasing. That the universe continually tends towards disorder.

~06:05Life is exactly the opposite of this. Life is actually maintaining extreme order in the face of the second law. And so all life must constantly battle entropy. And many have described life at the very highest level as being really the ability to maintain order, to fight entropy, which of course requires constant energy input. And metabolism is the process that all cells use to do this. Now for humans, I think we all know we need to eat and breathe in order to stay alive. For you future MDs out there, what happens if your heart stops, or you stop breathing? Well, of course, you die in a matter of several minutes. Why is that? Well, that's because every cell in your body has to do constant catabolism in order to derive energy, in order to remain viable. This means they need constant food, and particularly oxygen delivery. And if you don't get those things, metabolism can no longer work, and the cells die. And so energy is sort of this mystical concept that we like to talk about. And so before we delve a little bit deeper into what I mean by biological energy, what it is, why we have to constantly do catabolism to maintain it, I want to go back and say just a little bit more about carbohydrates and finish our discussion there, and talk about carbohydrates and sugar polymers, polysaccharides, because this has some additional properties I didn't have time to cover in my prior lecture that is important to understand how it allows these molecules to store energy in a very efficient way. And it will also enable us to talk a little bit about how carbohydrates can also be used as structural molecules for lots of different organisms. All right, so a little diversion to discuss carbohydrates and polysaccharides. And so if you recall from my prior lecture, sugars that are greater than four to five carbons can exist either in open chain or ring forms, furanose or pyranose rings. I'll just remind you by redrawing up here, glucose. So this is D glucose, drawn in the open chain form. Remember this can form a ring. OK. So it's a reminder, carbon 1, 2, 3, 4, 5, 6. If we have this hydroxyl group from carbon 5, form a hemiacetal bond with the aldehyde at carbon 1, you get this ring structure. This would be alpha or beta, depending on whether the hydroxyl group here at carbon 1 is pointing up or pointing down. D-glucopyranose. So this is review of what we already talked about last time. Now, what I want to talk about is that if we form a disaccharide or a polysaccharide-- that is, begin to make sugar bonds, and if we do that in a way that ties up this hemiacetal or hemiketal bond, this then prevents the ring from opening and gaining access to that carbonyl, that aldehyde carbonyl, as this moves between the open chain and the ring form. And so a good example of this is the disaccharide sucrose. So sucrose is a disaccharide of glucose plus galactose. And here is what this disaccharide looks like. OK, so you have here, glucose in the alpha-D-pyranose form. Here's fructose in the beta, because the OH group is pointing out. Fructofuranose form. And so the formal name for this molecule, sucrose, would be alpha-D-glucopyranosyl 1-2, because we're going from carbon 1 of glucose to carbon 2, there's 1, 2, 3, 4, 5, 6 of fructose. Beta-D-fructofuranose. Sucrose or alphaD-glucopyranosyl 1-2 beta-D-fructofuranose.

~09:14OK, so this disaccharide forms a bond between carbon 1 and carbon 2. That would be the aldehyde in glucose, or the ketone tied up in the hemiacetal or the hemiketal bond of these two molecules. And so there's no way that either of these sugars can access the open chain form, without breaking this O-glycosidic bond. And that's one reason why sucrose is a very good storage molecule for carbohydrates, because without breaking that bond, you prevent access to gaining these reactive aldehyde or ketone bonds, that could react with some other molecule in the cell. Now, I mention this because whether or not a particular sugar is enabled to get access to this reactive aldehyde or ketone really forms the basis for a classic sugar detection lab test. And this is relevant to discuss this lab test because it explains some nomenclature that is still used, and in fact, we will continue to use a bit as we discuss some aspects of sugar metabolism. So what this test is, is basically, is that if you heat the sugar with copper, which is blue, and that copper can gain access to a free carbonyl--that is, if the sugar can access the open chain form to expose the aldehyde or a ketone, that copper can then become reduced to copper plus. That changes the color from blue to red. And so if you reduce the copper, you oxidize the bond there. And this test turns positive if there is a reducing sugar, is what it's called. So a sugar that reduces the copper from the blue 2 plus state to the red plus state is a positive test. And so basically, a reducing sugar is any sugar with an ability to access an open chain form. Because that will provide the free aldehyde or ketone to reduce the copper in this test. So this term, reducing sugar, obviously applies to all monosaccharides, because every single one of them has an aldehyde or a ketone, and can access the open chain form. But it will only apply to some disaccharides or polysaccharides. So let's come back to sucrose up there. So is sucrose a reducing sugar? No, it's not, because there is no way that you can access a free aldehyde or a ketone in sucrose, because it's tied up in that O-glycosidic bond between carbon 1 of glucose and carbon 2 of fructose. And so you need to break that bond to give them monosaccharide subunits in order to access the open chain form. However, let's give an example of a disaccharide that is a reducing sugar. And that's the disaccharide maltose. So maltose is basically two glucose molecules. And those two glucose molecules are like this. So this has a O-glycosidic bond between carbon 1 of this glucose, and carbon 4 of that glucose. And so formally, this would be alpha-D-glucopyranosyl 1-4 alpha or beta. If it's pointing down, it's alpha, pointing up, it's beta. Dglucopyranose. So maltose or alpha-D-glucopyranosyl 1-4 alpha or beta D-glucopyranose is a reducing sugar, because this sugar could access the open chain form. You could open up this carbonyl and expose the free aldehyde at position 1. Now, we could also refer to this sugar as having two different ends. We can refer to this end as being the nonreducing end, and this end as being the reducing end. Because of course, this bond, this aldehyde on the first glucose is tied up in this O-glycosidic bond, whereas the one on this one is not. So this end is non-reducing, and this end is reducing. Now this becomes much more relevant if we start talking about polymers. And so what starch is. So starch, what potatoes are made out of, is basically a polymer of maltose. Polymer of maltose molecules. So basically our glucose with 1-4 linkages.

~12:39OK, so I could draw that end, the reducing end of the polymer, in the alpha or beta. But this here, being the nonreducing end. Every other subunit is tied up in this 1-4 O-glycosidic bond. And so starch is really a polymer of glucose molecules with bonds between the 1 and the 4 position of each subunit. Now we will see later that we build and break down starch polymers from only one end, from the non-reducing end. And so having these terms, reducing and non-reducing, provides the term to specify different ends of a polymer. Reducing and non-reducing ends turns out to also be important for naming conventions for disaccharides or polysaccharides. And this becomes relevant if we consider the disaccharide lactose. So lactose is also a polymer of glucose plus galactose, except this polymer of glucose plus galactose, unlike sucrose, is different because it has a different linkage between the sucrose is a disaccharide of glucose plus fructose. Fructose, I wrote the name correctly. But lactose is a disaccharide between glucose and galactose. And so I'll draw it here. OK, so this is lactose. This is galactose here on the non-reducing end. So the formal name for this would be beta, because there's a beta linkage here between carbon 1 of this galactose and carbon 4 of this glucose. So be beta-D-galactopyranosyl 1-4. And this is glucose in the alpha or beta. D-glucopyranose. Lactose, or beta-Dgalactopyranosyl 1-4, alpha or beta, depending if I drew down, alpha, up, beta. D-glucopyranose. Now, this also has non-reducing and reducing ends. And by convention, you would name the sugar from the nonreducing end to the reducing end. Hence I put galactopyranosyl 1-4 alpha-D-glucopyranose, named them in that order. Now you'll note that lactose, unlike starch, unlike maltose, unlike sucrose, links these disaccharides with a beta linkage between this sugar and that sugar, where the other ones had alpha linkages. And structurally, this is very different. And I just want to point that out. So this here is basically alpha-D-glucopyranose. And so alpha, this is carbon 1. This is the hydroxyl pointing down. That's why it's alpha. If I make an O-glycosidic bond to another sugar, you can see that it points and creates this kinked structure. Now, if I were to make this beta, this hydroxyl rather than being here would be at this position on carbon 1. You can see that that now creates a very different geometry. Now you have a flat molecule, as opposed to a kinked molecule. Now, this has consequences, of course, for the enzymes that break down the sugars. Obviously, it's going to be a very different enzyme that breaks a bond in this orientation, versus a bond in that orientation. This has, basically implications for what enzymes we have. And so lactose, of course, is milk sugar. All mammals make milk, so make lactose, and break it down when we're babies. But most mammals tend to lose expression of the enzyme lactase that's able to break this 1-4 beta linkage as we age. And this is basically what accounts for lactose intolerance. Now, if you think about it, in the world, much of the world has issues with varying degrees of lactose intolerance in adulthood. The exception to this is often people of European ancestry. Why is this?

~15:36Well, Europeans historically drank milk well into adulthood. And so this selected for continuous lactase, the enzyme that wouldn't break that beta 1-4 linkage. The expression as people move into adulthood. And so it's an example of how cultural things, drinking milk into adulthood, really affected evolution, such that that subpopulation of people, people with that genetics to increase lactase expression, don't become lactose intolerant as they age. Now, this alpha versus beta linkage-- that is, whether or not you have this more kinked versus more flat structure-- also has a major effect on the structure of a polymer as well. And so if we look here at this starch polymer, it's basically this series of glucose molecules stuck together with alpha 1-4 linkages. So that's going to create a kinked structure. This is best shown here on this slide. So you can see this kinked structure. And so when you build this polymer, you're going to end up more with this helical structure in 3D space. That's a very efficient way to store glucose monomers in much less space than you would otherwise get with beta linkages. Now, it turns out that nature takes this a step further. And in addition to just having this starch polymer, it turns out sometimes you can add branches to the starch polymer to further increase the efficiency of energy storage. So if you have here a starch polymer, so let me just draw here a couple subunits. So this here is a starch polymer, glucose polymer with alpha 1-4 linkages that I can put an additional branch on this by adding on a linkage up here on the top. With an alpha 1-6 linkage. So the 1 position of here, to the 6 position of here, which has the effect of creating branches on this long polymer, such that you have a single reducing end, and lots of non-reducing ends. Each of these non-reducing ends has a polymer of alpha 1-4 linkages that would be stuck to the main chain polymer via this alpha 1-6 linkage. And this has a couple implications for storage. The first is, by only having one reducing end, it reduces maximally the exposure to a free aldehyde. And so that makes it a good storage sugar. And also as we will see, we're going to break down these sugar polymers biologically from the non-reducing ends, and build from those ends. And so there's lots of hooks or lots of places to either add or remove sugars from, which allows you to access carbohydrates quickly as needed. That's shown nicely here on this slide, better than I can draw it. So up here, you have your non-reducing end across the polymer. So this here would be starch with the alpha 1-4 linkages on the polymer, or reducing end on one side, non-reducing end on the other. You can then add branch points to that by making an O-glycosidic bond between carbon 1 down to carbon 6, creating a branch. And so you can see this helical branch structure will then form. That is both very efficient to pack lots of glucose into one place, and give lots of non-reducing ends with which to build and remove sugars from for biology to either store or quickly access sugar molecules as needed. Now, in plants, of course, potatoes use starch in a straight chain. We know that. However, plants also use this branched structure. This is a molecule called amylopectin. Amylopectin is better known in the food industry as Sure-Jell. It's the material that allows you to make jelly. It's the jelling agent in jelly. And effectively, this has every 24 to 30 units. There would be a branch with one of these alpha 1-6 linkages to give you this multi-branched structure.

~18:57Now, animals don't make starch. Animals don't make amylopectin. But animals make another molecule called glycogen. Glycogen, also a sugar polymer, exactly like amylopectin. Alpha 1-4 linkages with the alpha 1-6 to create branch points. Just like amylopectin, except glycogen has even more branches with a branch every 8 to 12 glucose units. And so, this is a very complex structure for both plants and animals to quickly store and quickly release glucose molecules when they're needed. Now, this is in contrast to a glucose polymer that can be done that has a very different 3D structure. So what if we take starch, and rather than have these alpha 1-4 linkages, but instead replace these with beta 1-4 linkages? OK, so now beta linkages. Looks simple enough, but changing the geometry from the alpha to the beta bond removes the kink. Now it's much more of a flat structure. Turns out this is, shown on an image here. So here's a beta linkage, and suddenly you have this flat structure. This is the same polymer as starch chemically, but has a different linkage, the beta 1-4 linkage. And that polymer with the beta 1-4 linkage is cellulose. And cellulose is, of course, what wood is made out of. And so wood and potatoes, cellulose and starch, exactly the same polymer. Same number of calories, if you could access the glucose units. But the alpha and beta bonds make them very different. Obviously, wood and cellulose is a very good structural polymer for plants. We build houses out of it. We don't build houses out of potatoes, but we eat a lot of potatoes. So equally good molecule for plants to store lots of food for energy. Turns out wood is a great source of energy too. You just need to have an enzyme that can break the beta 1-4 bond. Termites have symbiotic microbes that allow them to do this, and this is why termites can eat wood, a lot of energy tied up in wood. Now, we lack the time to go into the details about how this relates to other structural carbohydrates. But in general, structural carbohydrate or carbohydrate-like molecules form polymers of sugar or related molecules, also with these beta linkages. And a good example is chitin, so the material in the insect shells, is basically a sugar-like molecule that's a polymer with a beta linkage in it. Same thing with cartilage in humans and animals. And you can look up the details of what these things look like, of course, if you're interested. Chitin and cartilage aren't made of true polysaccharide polymers, but they're very related to polysaccharides. And they really illustrate how nature can take carbohydrate chemistry and repurpose it to basically act as an energy source, but also build all kinds of structural molecules that are really useful in biology. All right, now I want to go back to, here's ways that one can store and use carbohydrates in interesting ways. But now I want to come back to them as energy sources, and assuming you can access the glucose and whatever polymer it's present in, how can you actually metabolize it to provide energy in a way that sustains life? And this means we're going to turn to another topic, a topic that's typically referred to as bioenergetics. Bioenergetics, which is really the discussion of how energy is transduced in biological systems.

~21:58I'm going to introduce this topic today, we're going to revisit it throughout the course. But it's really important to ask this question. We really want to consider, what do we mean when we say biological energy? What is biological energy? Well, someone probably made you memorize in high school, and often you were thinking, oh, it's just ATP. And certainly, ATP, adenosine triphosphate, is a very important molecule for biological energy transduction. But I want to actually explore why ATP is actually useful. And if ATP is energy, why don't we just eat all kinds of ATP? I can say for a fact that absolutely none of you had ATP today for breakfast. No one sells ATP as an energy booster. And so if ATP is such a great energy source, why don't we just eat it? And to really understand ATP, how it works, what biological energy really is, we really need to revisit some very basic topics in thermodynamics.

~22:50Now, thermodynamics strikes fear into the hearts of students everywhere. You can get details of thermodynamics and the theory around these things in other courses. That's not really what we're going to try to accomplish here in 705. Our goal here is really a practical understanding of how thermodynamics applies to biology and metabolism. And we need to go into this, because it's key to understanding why we store energy, go through all this trouble to store energy as these carbohydrate polymers to begin with. Why we eat potatoes and not ATP, as well as why ATP is actually useful to cells. And so let's take a step back and just think about it. Let's think about wood. I just told you, wood is a polymer of glucose molecules with beta 1-4 linkages. How can we, forget as organisms, as cells. But how can you release energy from wood, to do other kinds of work? Well, we can burn it. And so what's the chemistry of burning wood? Well, it's a carbohydrate polymer. So it has this CnH2n formula. If we combine that carbohydrate with oxygen, it releases CO2 and water, plus some light, plus heat. This light and heat is energy, because we can use it to do work. Boil water, turn a turbine, make electricity. Whatever. Warm ourselves by the fire. Use the light to do some other things. And so this burning of wood is certainly release of energy. Now, we lack the enzymes to do this reaction in wood, because we can't break that beta 1-4 linkage. But we certainly can break that alpha 1-4 linkage in starch from potatoes. And we use the exact same chemistry to burn that glucose and release energy. Except the difference is that if I burn wood, I do this all in one step. But life is a much better engineer than that. It basically, rather than releasing all of that energy in one step, it really releases it stepwise in a manner that's actually useful to cells. But it's exactly the same release of energy. And so what life is, or extracting biological energy is, is it's really the ability to do stepwise oxidation of glucose or other carbon. I'll write glucose for now. To get energy. So we burn wood, that's favorable, it releases energy. If we do stepwise oxidation, we also can release energy as well. Same reaction, also releases energy.

~24:59How much energy do we get from releasing us burning glucose, as you do and burning it in a fire? Well, it has to be exactly the same amount, because it's exactly the same reaction. So we burn wood, we get light and heat. Cells burn glucose, it releases the exact same amount of energy. It just does so in a way that allows the cells to do biologically useful things. What are those? Well, it could be heat. All of us maintain temperature. So heat is a conversion of energy that of course, is useful to biology. But it can do other things, too. It can allow cells to move. It can allow them to do any reaction to fight that entropy that allows life to exist. And so all things, including ourselves, have to follow the same laws of thermodynamics. Life is not special in that regard. And so burning wood is favorable, because it increases the entropy of the universe. You're breaking up this polymer into a bunch of monomers. That's going to be spontaneous. That is in accordance with the second law of thermodynamics. True if cells burn it as well. But remember, the second law says that net entropy of the universe must always increase. And so if we're going to do something that's not energetically favorable, like build a glucose polymer, we obviously have to put energy into it. And to do so, the energy that's released must be greater than the energy that we actually store. And so this is actually a really key point, because anything that we do that requires energy input for a cell to carry out that process requires a source of energy release somewhere else that is equivalent or greater than what is actually put in. And so cells need energy, because they do lots of thermodynamically unfavorable things, fighting that entropy of the universe. And all of those processes must come from the release of energy elsewhere, like the burning of carbohydrate. And so that constant energy input is at the very highest level, why we need to do constant metabolism in order to maintain order and survive as organisms. Now this, of course, comes from things like burning of carbohydrate. But of course, that carbohydrate has to come from somewhere. And so ultimately, you have to have an external source. That external source is of course, the sun. Photosynthetic organisms can use light energy from the sun to do exactly the same things. Build those excess polymers, and that's why we as animals or anything that eats other animals as a way to live is ultimately depending on eating photosynthetic organisms, because photosynthetic organisms do this by harvesting energy of the sun. Now that's all very high level. But I want to come back to the specifics of metabolism, ultimately, and understand those as how they relate to enzymes' reactions and pathways. That is, how do you get that complex series of reactions that make life possible, and ultimately still be guided by these exact same principles? In other words, how do we couple the energy releasing reactions, like burning glucose, in a way that obeys the laws of thermodynamics and operates under biologically acceptable conditions in order to do things that are biologically unfavorable. That's really what bioenergetics is. And so the next thing we want to consider then, if we want to understand this, is really, let's ask the question. What determines if a specific reaction occurs? That is, what determines if something like burning wood actually spontaneously happens?

~28:08If you light wood on fire, it will burn. It does so every single time, everyone has had that experience. But no one has ever seen CO2 and water spontaneously come together and form a tree. That doesn't happen. So what determines whether you burn wood and that is favorable, versus why it is that CO2 and water don't come together and spontaneously form a tree? Do not think that this is determined entirely by enzymes. Enzymes are catalysts. Enzymes are the spark that makes the burning of wood possible. But remember, enzymes cannot change thermodynamics. Enzymes only change the rate at which a reaction happens. It does not change the equilibrium. That tree wants to, if given the catalyst, form CO2 and water. That reaction is spontaneous because it is thermodynamically favorable. That tree will not take CO2 and water and spontaneously reform a tree. And there's no amount of enzyme that will make that happen all by itself. And so remember that. If you have any reaction between A and B, there is some equilibrium, as defined by the chemical properties of A and B, such that the equilibrium lies far to one side or the other. And that equilibrium is determined by thermodynamics. It is not determined by enzymes. I don't care how much enzyme I add. If the equilibrium lies towards A as I draw it, I can add all the enzyme I want, and I can never create more B. And so, this is super important, and is actually a point that is gotten wrong by lots of biologists. They think, oh, this enzyme is expressed. Therefore, this pathway must be happening, or this reaction must be happening faster. You need an enzyme to catalyze a reaction. So a reaction may not occur, your wood may not burn unless you give it a spark, a catalyst to actually make it happen. But you cannot add a catalyst to fight thermodynamics. You cannot take that CO2 and water and turn it back into a tree. And the same thing is true. You can add all the enzyme in the world you want, and all it will do is help A and B establish the equilibrium that is defined by the thermodynamics of the relationship between A and B. Now, what determines the equilibrium between any two species? That's a topic for another class, why a species is favored to be one side or another. However, a useful tool to think about this in biochemistry and quantify this for any pair of reactions is something you've probably learned about before. It's something called the Gibbs free energy. Gibbs free energy, or delta G. Delta G is related to the way we'll talk about the equilibrium constant. And it's basically a term that we can use to specify if a reaction is favorable or not. And so hopefully you learned in an introductory class that if delta G is less than 0, our reaction is spontaneous. If delta G equals 0, our reaction is that equilibrium. If delta G is greater than 0, our reaction is not spontaneous. All right, if we take our log and ask, what is delta G to turn that log into CO2 plus water, it is less than 0, because it is very spontaneous. We take a pile of ashes and ask what delta G is to recreate the log. It's greater than 0, because it ain't going to happen. And if it's at some equilibrium, delta G is equal to 0. Now, delta G depends entirely on conditions, as we will see in a minute. And because of that, this means that there can be conditions where absolutely any reaction can be favorable if the conditions are right. And we'll see that this becomes very relevant to understand how metabolism works. And so let's go back and just now consider some generic reaction, A and B.

~31:37So at equilibrium, what happens? So at equilibrium, the concentration of A and the concentration of B are not changing. Delta G equals 0. These two things are at equilibrium, whatever that equilibrium is. A and B are not changing in concentration. Now if I come and I add more A to this side of the equation, what does that mean? Well, you know from Le Chatelier's principle that that's going to favor production of B. We have shifted things now out of equilibrium. So if I add A, that leads the production of B to re-establish this equilibrium. That means that delta G from A to B is less than 0 until I've reestablished equilibrium, and now delta G is back to 0 again. Now, what happens if I add B? Well, if I add B, now I'm going to produce some A. So add B. That leads the production of A, until I reestablish that equilibrium. So delta G, in that case, to go from B back to A. The reverse reaction is less than 0. Or I could also say that delta G, to go to A to B here, because that's not going to happen. I just added more B. I'm not going to suddenly create more B from A. So here now, delta G is greater than 0. Now note when I did this, I don't actually have to specify how much A or how much B I've added. The equilibrium depends on the ratio of B to A. Not the absolute concentration of either species. And so having a way to think about this equilibrium, it turns out, is also useful for biological systems to divine some delta G that is helpful to relate to this equilibrium constant. And that is this concept of delta G 0 prime. A standard free energy, which for biological systems, relates to the equilibrium constant at 25 degrees, or the pH of 7 and 1 atmosphere. Pretty typical biological conditions. And this here is basically related to the equilibrium constant. And we can calculate the actual delta G by the following formula. Delta G equals delta G 0 prime plus RT times the log of the products over the reactant. So this would be drawn for the reaction A to B. So the reaction A to B is related to delta G 0 prime, which is related to the equilibrium constant. I'll tell you how in the second. Plus R is the gas constant, T is the temperature in Kelvin times the natural log of the ratio of products, B over reactants, A. This tells you whether or not at specific concentrations of B and A, that is a specific ratio of B and A, whether it is favorable to go from A to B, delta G less than 0, or whether it is favorable to go from B to A, delta G greater than 0. And so this should begin to make it clear that whether or not a specific reaction occurs is affected by the actual conditions present. And you can calculate that based on this relationship. And so if I specify concentrations of A and B, as well as delta G 0 prime, I can know, at those concentrations, which direction of the reaction is favored. Now of course, my drawing A to B is entirely arbitrary. I could equally draw it B to A. And if I did that, so this is delta G 0 prime, A to B, all I do is flip the signs. So delta G equals delta G 0 prime. The negative of A to B is equal to the positive of delta G 0 prime from B to A, because the direction is arbitrary. Plus RT log in this case. Now I have A is my product, and B is my reactant. If I just flip the sign, flip that ratio, it's just going to change the sign of my product and give me exactly the same result with the opposite sign, which makes sense. What's favorable in one direction is not favorable in the other direction, and vice versa.

~35:17Now, I can also set delta G equals 0. That's equilibrium. If I do that, then delta G 0 prime equals negative RT. So this would be for A to B. I'll use the top example. So delta G equals 0. Then I have delta G 0 prime equals RT times log B over A. So if I know two things are at equilibrium, I can calculate delta G 0 prime, and know what the equilibrium constant is. And so it follows from this then that if delta G 0 prime is negative, that means B is favored over A at equilibrium. And if delta G 0 prime is positive, that means A is favored over B at equilibrium.

~35:55And so delta G 0 prime really is a convenient way to look at a reaction, and know which direction the equilibrium lies. So A and B, delta G 0 prime is negative, equilibrium lies to B. Between A and B, if delta G 0 prime is positive, the equilibrium lies towards A. But the key concept is that for any specific reaction, any specific conditions of A and B, whether or not A is converted to B will be defined by the equilibrium constant. But it will also be defined about what delta is under those conditions. So to be clear, delta G will depend on conditions, because I'll write it again, delta G equals delta G 0 prime plus RT times the log for the reaction, A goes to B. So products over reactants. And so whether or not A is converted to B will be a property of the equilibrium constant, plus the concentration of B and the concentration of A, that ratio of concentrations at the conditions present. And if in that calculation, delta G is less than 0, that reaction is spontaneous. If delta G is greater than 0, that reaction is not spontaneous. Or to put it another way, if delta G is less than 0 under those conditions, energy is released. Or if delta G is greater than 0, that reaction will not occur without energy input. If I burn wood, energy is released. But there's no way I can reassemble that log without some kind of energy input. And really getting this concept is central to understanding metabolism, as well as what biological energy means. And hopefully, what is now apparent, based on what I just said, is that what really determines delta G is two things. Of course it's the equilibrium constant. But in essence, delta G for any reaction is proportional to the ratio of the reactants over the products. And that I can come up, at least in theory, with any relationship, any ratio of reactants to products to make this favorable. At some tiny, tiny concentration, if I have nothing but CO2 and water, and infinite time and infinite catalysis, a tiny bit of wood could spontaneously form. And so life really creates the conditions that allow that, and select for that to happen, and of course, add energy input so it can actually happen more. And so this should be very clear to you that absolutely no reaction is irreversible. I don't care what your textbook says. Lots of sources will discuss irreversible reactions. I'll talk about irreversible reactions later in the course. But what we mean when we say the word irreversible reaction is these are under conditions that are found in cells and in nature. And I stress this because if we want to understand the energetics for how pathways work, we have to appreciate that life can create conditions to do unfavorable things, because that's the only way that pathways can work.

~38:33All right, what what do I mean by that? Well, suppose again, we'll come back to our reaction. A goes to B. Suppose I need to build a pathway where I need to convert A to B, because B is actually useful for some purpose that I need to do in a cell. However, what if this is at least by equilibrium, unfavorable? What if the equilibrium lies towards A? That means delta G 0 prime is positive, is greater than 0, for A to B. What does that mean? Delta G 0 prime greater than 0, for the reaction A to B? That means the equilibrium lies towards A. And so there is no amount of enzyme I can add that will allow me to net convert A to B, because the equilibrium lies far towards A. However, whether or not a cell is able to build a pathway, where it converts A to B depends on delta G, not the equilibrium constant for that specific reaction. So how can I turn A into B? Well, I could turn A into B if I keep the concentration of B low enough that delta G, not delta G 0 prime, that's the equilibrium constant, but delta G favors A to B conversion? That is, delta G for A to B is less than 0. How can I do that? Well, remember delta G equals delta G 0 prime plus RT log of B over A. So this term is positive, because I told you, the equilibrium lies towards A. So I need a concentration, a ratio of B to A, such that B is low enough that this term is more negative than that term is positive. And if that's the case, delta G will be less than 0, and I can net move that forward. So how can I do that? How can I keep the concentration of B low? Well, I can build a pathway that consumes B in a way to keep it low, which favors A to B conversion. In other words, I can build a pathway, A goes to B goes to C. So even here, where delta G 0 prime is greater than 0, if delta G 0 prime for this reaction, B to A, is much less than 0, such that equilibriums strongly favor C, that means my unfavorable equilibrium here can be overcome by the very favorable equilibrium of B to C. In essence, I can use B to C to pull A to B, such that it actually happens. Produce this intermediate that's useful on the path to making C. This strategy is used in lots of metabolic pathways. And it's useful to generate lots of chemically useful intermediates. Now note that this only will work if conversion of A to C is favorable. That is, if delta G 0 prime is less than 0. I can only pull this reaction if delta G 0 prime is less than 0. That is, if equilibrium favors A to C, I can build a pathway this way to do something unfavorable on the way to making C. This brings up another key point, in that everything that we discuss, relationship between any two metabolites in any single reaction, also must be true for entire pathways. So I have a three-step pathway from A to C. Whether I turn A into C in one step, or turn A into C in multiple steps, the free energy is exactly the same. If I burn wood by lighting it on fire, it releases the same amount of energy than if a termite has an enzyme that breaks that beta 1-4 linkage in the wood, and can burn that glucose stepwise through metabolism, the exact same amount of energy is released. Glucose on one side, CO2 and water on the other side, exactly the same amount of energy released, whether I do it in one step, or I do it in multiple steps. The equilibrium constant between those has to be the same, and the delta G has to be the same between all of it.

~42:13And so it should become apparent, then, that A to C has to be favorable if I'm going to use this trick of keeping product low in order to pull an otherwise unfavorable reaction forward. In other words, there's no way for me to keep B low and net convert A to B all on its own. That will not work. And it's these unfavorable reactions that ultimately then require energy input. And this is where ATP now starts becoming useful for cells. OK, so now I want to talk a little bit about how ATP provides energy that allows otherwise unfavorable reactions to occur. And ATP is useful because delta G applies to sets of reactions in the same way it applies to single reactions. That is, it applies to whole systems. All reactions are a series of reactions, whether they happen alone or coupled together. All have to follow the same rules. So now let's make our reaction more complicated. Let's say A plus B goes to C plus D, where this is really two reactions coupled together. A being turned into C, and B being turned into D. All right, so will this reaction happen? Can I turn A plus B into C plus D? Well, how do I know? Well, it's going to be, got to calculate delta G. And so it's going to be delta G 0 prime for A going to C, plus delta G 0 prime for B going to D. So those will relate to what's the equilibrium constant, the equilibrium relationship between A and C and B and D. If it's less than 0, equilibrium will favor to the right. If it's greater than 0, equilibrium will favor to the left of A and C and B and D. Add them together, that gives me my overall delta G 0 prime for those two reactions. Plus RT times the log of the products. So that's C and D over A and B. Products over the reactants. So if I take an unfavorable reaction-- say, A to C, if delta G 0 prime is greater than 0, so it would favor A, and I couple it to another reaction, B to D that's very favorable, because I add these two terms together. I can now take something where the equilibrium would be unfavorable, and make it favorable. However, whether or not this actually happens still depends on this ratio of products to reactants. So it still depends on the ratio of C to A, and D to B in our hypothetical thing here. So to know if a specific reaction will actually occur, we have to take into account both the sum of the delta G primes related to the equilibrium constant, and the actual ratios that are present in cells. And so the net effect is that I can couple more favorable reactions to make otherwise unfavorable reactions now become favorable. That is, have energy input. But it still depends on conditions. And so let's think a little bit about the polymer synthesis that we learned from Professor Yaffe. And so in that process, what did we do? We made several polymers, and you learned about the chemistry to do this. We made DNA, we made RNA, and we made protein. These are polymers of nucleic acids. Protein is a polymer of amino acids. All of these fight entropy, right? We're building polymers, not breaking them down. And all of them were synthesized using reactions that hydrolyzed ATP. In fact, those reactions actually hydrolyzed ATP in the following way. And every single one of those cases, if you look back, what you will see is that they all had steps where ATP was taken to AMP plus 2 inorganic phosphates. This reaction is very favorable. In fact, it's two reactions. It's really ATP goes to AMP plus pyrophosphate.

~45:39And then that pyrophosphate goes to 2 inorganic phosphates. It turns out that delta G 0 prime is less than 0 for both of these reactions. That means the equilibrium lies towards AMP plus pyrophosphate. And here, the equilibrium lies towards 2 inorganic phosphates. And so there's actually two tricks here by metabolism. One is, it's two very favorable reactions that we're coupled to do something unfavorable, build a polymer. And use the trick of keeping pyrophosphate low by coupling it to a downstream, very favorable reaction, which even further pulls this reaction, and is why ATP hydrolysis here becomes so useful as a way to build these polymers. Let's just go quickly and add a few numbers to show exactly what I mean by how ATP hydrolysis can be useful in this setting. And so, let's do something a little simpler. Let's go to ATP plus ADP plus to inorganic phosphate. So let's give this some numbers, delta G 0 prime in this case is equal to negative 7.5 kcals per mole. All right, what does this mean? Delta G 0 prime is related to the equilibrium constant. It's less than 0, which means the equilibrium lies towards ADP plus phosphate. Now let's see how this actually helps by considering what the first step in glucose metabolism by cells is. So the first step cells do in glucose metabolism is add an inorganic phosphate group to glucose. So it's this reaction, glucose plus inorganic phosphate goes to glucose phosphate. Just a quick note on shorthand that I will use in this course. So PI, of course, is inorganic phosphate. That is this PO43 minus group. When it forms a phosphodiester bond, like to an alcohol and one of the glucose molecules, I'll draw this in a future lecture. I'll oftentimes indicate it by putting a circle around the phosphate. And so adding this phosphate to glucose is a step that traps that glucose in cells. For now, let's just consider this reaction. And so delta G 0 prime of this reaction is positive 3.3 kcals per mole. What does that mean? Delta G 0 prime relates to the equilibrium constant. It says the equilibrium constant lies here to the left, to the glucose plus phosphate side. So if we want to net trap glucose phosphate in cells, well, it's not going to happen spontaneously. You need some kind of energy input. That can come from ATP. And so if we couple those two reactions as follows. So we now have glucose plus ATP goes to glucose phosphate plus ADP. Now let's draw this out. Will this happen? So we can calculate delta G. Delta G equals delta G 0 prime. So there's two reactions happening here, glucose to glucose phosphate, and ATP to ADP. We know what delta G 0 prime is by adding those together. So it's 3.3 plus negative 7.5. That equals negative 4.2. What does that tell us? That says, now we've created conditions where the equilibrium constant lies towards the right, towards the glucose phosphate plus ADP side. But how much of this occurs or whether it occurs isn't just the property the equilibrium constant. It's also how much is there. And so that's RT times the log of the products, glucose phosphate.

~48:34And ADP over glucose. And ATP. And so, if this term is less than 4.2-- that is, if the ratio of glucose phosphate to glucose and ADP to ATP in this term ends up being less than positive 4.2, this reaction is spontaneous. It is favored. Lots of conditions where that would be the case, although if this term is 4.2 or greater, now it will no longer occur. How much ATP is needed? Well, it's hard to answer this question in absolute terms, because if there's a lot of ADP around, you need a lot more ATP for the ADP to be useful. And this is the problem with equating ATP with energy. The energy here is in the ratio of ATP to ADP, or ADP to ATP. And this is something that we we'll come back to in the next lecture, because in the end, this is what will determine if a reaction has happened. It is not the absolute concentration of ATP. It's the fact that ATP hydrolysis is favorable. But how favorable that is depends on the relevant concentrations of ATP or ADP. And relative concentrations are all that matters in thermodynamics. It is not absolute concentrations. It is the ATP-ADP ratio that is the correct way to think about energy. And this is true for any reaction with respect to energetics. If an ATP-ADP ratio exists, such that when coupled to a reaction, delta G is less than 0, then it becomes spontaneous. And this is how ATP provides energy. And we will come back to this more in the next lecture. Thank you.

🎬 MIT 7.05 Lec 20 · Bioenergetics & Glycolysis I (Prof. Vander Heiden)⏱ ~50:00

~00:00MITOCW | 20. Bioenergetics/Intro Pathways/Glycolysis I PROFESSOR:

~00:02Today we're going to discuss bioenergetics, continue that discussion and use that as a transition into also beginning to see how that applies to understanding glycolysis, the pathway that allows glucose breakdown. So just as a reminder of a couple of the points that we discussed last time, so last time we discussed what makes reactions favorable. And so remember, you could have any reaction, A to B-- A to B could be a single step. A to B could be an entire pathway, all steps in the pathway, that whether or not that reaction or pathway happens is determined by thermodynamics. It's not determined by enzymes. Enzymes are important, of course, to control the rate of reactions so they may allow things to happen that otherwise wouldn't happen. But whether the reaction happens or not is determined by free energy. And so free energy, delta G, remember we discussed is the following relationship. So it's related to this constant delta G0 prime, which is related to the equilibrium constant for any pair of products and reactants as well as this formula rt times the log of the products over the reactants. And so remember this means that whether a reaction happens depends on the equilibrium constant, but it also depends on the actual conditions present. That is how much substrate and how much product is there. And so what this means is that biology can come up with ways by keeping the product concentrations low to do things that may move in the opposite direction of what you would predict from equilibrium. And also by coupling these less favorable reactions to more favorable reactions, you can also allow biology to do unfavorable stuff. And this is, of course, where the role of ATP comes in. And we discussed this at the end of last time as well. Of course, what's important then becomes the ratio of ATP to ADP. And to illustrate that, we use this example. We use the-- remember ATP hydrolysis has a delta G0 prime of minus 7.5 kcals per mole. That, of course, tells you this is about the equilibrium constant. That tells us the equilibrium lies to the right. We can couple it to this first reaction in glycolysis, which traps glucose in the cell by adding a phosphate group. Delta G0 prime for this is positive 3.3 kcals per mole. This positive number tells you that the equilibrium lies towards the left. But when we couple these two reactions together, we add these two numbers together, and now we get a delta G0 prime of minus 4.2 kcals per mole, which are the coupled reaction. The equilibrium lies now again to the right. And that's how we can use ATP hydrolysis in order to carry out this unfavorable process, trapping the glucose in the cell.

~02:38Now how much glucose will be trapped? Well, of course, that will come from this reaction up here. And that's because whether or not a reaction occurs or not of course depends not on the equilibrium constant but on the actual conditions and the delta G of that reaction.

~02:54And so we can just plug that in for this system. And so we see delta G0 prime minus 4.2 kcals per mole. That says equilibrium lies to the right. But how far really depends on the conditions. And that's defined by this ratio of products over reactants. And you can imagine that this term here, as long as it's less than 4.2, you will favor glucose trapping inside the cell, whereas, of course, if it's greater than 4.2, then you'd favor-- the opposite direction would be favorable. Now this is really critical because this really explains how it is that ATP actually works to help cells do various reactions. And so if an ATP-ADP ratio exists such that it can make delta G less than 0 for the reaction that it's coupled to, that is how it provides the energy that then allows unfavorable reactions to occur. And so please do not make the common mistake that many, many biologists make when they state, oh, ATP equals energy. There's a lot of ATP. The cell must have a lot of energy. Remember it's the ratio of ATP to ADP that provides the energy. And so that means ATP can be sky high, but if ADP is equally high, there's no energy there. Consequently, ATP can be very low. But if ADP is extremely low, now you still have a high ATP to ADP ratio. And that is what provides the energy. And that's because this occurs regardless of concentration. It's because it's this ratio of ATP to ADP that ultimately says how much energy is there for the ATP to drive a reaction. In other words, delta G, or whatever the reaction is coupled to, is going to be proportional to the ratio of ADP to ATP. Great. Now how much ATP do you need then? What ratio do you need to drive a reaction? Well, it's hard to say that in absolute terms because it's going to be defined by how that ratio is coupled to the ratio of whatever other reactants and products. It also tells you, remember, as we talked about before, we can switch the directions of these reactions and we just flip the signs. The directions here are arbitrary. And so that means it also tells us how much energy we need to put in if we're going to charge up an ATP-ADP ratio. In other words, if we want to synthesize ATP, that is take ADP plus phosphate and make an ATP, well, what's this? What we just-- the exact opposite sign of that delta G0 prime is going to now equal plus 7.5 kcals per mole. And so that means that the equilibrium of this reaction is going to lie to the left just as that one lied to the right. And it's going to lie to the left by that much. And if we want to know how much energy it then takes to make ATP, well, obviously, it's not going to happen because equilibrium favors this direction. It's not spontaneous in that direction. But at various conditions, we can know by plugging in our formula delta G equals delta G0 prime plus 7.5 kcals per mole plus RT times the log of ATP. So if I have my ADP-- I'm sorry-- my ATP concentration sufficiently low relative to my ADP concentration, I can still make this reaction happen. Now that's not going to give you an ATP-ADP ratio that is actually consistent with being able to do anything else into the cell. And so but what it says is that if I am able to couple energy from nutrient metabolism to this reaction and overcome this positive 7.5 kcals per mole, that basically will allow me to now synthesize ATP and maintain an ATP-ADP ratio in the cell that allows cells to carry out other functions by the same rules that we talked about that would be unfavorable. Now there's lots of implications here for metabolism, because it says how much input you need to synthesize ATP. How much energy you need to synthesize ATP is going to depend on how charged this ratio is. And how useful that ratio is to catalyze other reactions is also going to depend on how charged that ratio is.

~06:50And in essence, this is why we cannot store energy as ATP. This is why we didn't eat ATP for breakfast and instead ate cereal that has a bunch of glucose in it, because we can use that glucose to get ATP but the ATP itself is not something that we really can burn to get energy in a sustained way. In other words, the delta G of ATP synthesis, making ATP is going to be proportional to the log of the ratio of ATP over ADP for all the reasons I described. The delta G of using ATP, ATP hydrolysis, using ATP to drive reactions is also proportional to RT times the log, in this case, of the ADP to ATP ratio. And so because you need to consume this ratio to get energy, and you need to put in energy to create this ratio, you are constant-- and how much is an exponential property-- you are constantly having to generate ATP in a way that allows you to then have a ratio that can then be consumed to fuel other reactions. And that's what's shown here on the slide. So I just graphed here what the ATP-ADP ratio is relative to a theoretical delta G that's shown here as a linear scale, here on a log scale, that basically there's this logarithmic relationship and so you need an exponential increase in energy input to drive this ratio up higher in order to, quote unquote, "store" more energy that can be used for later. And so this system is not one that you can use as an energy storage system and ultimately explains why cells have to do constant metabolism, have to constantly do some sort of metabolism to keep this ATP-ADP ratio charged for it to be in a useful range to have an ATP-ADP ratio that can be coupled to drive otherwise unfavorable reactions in our cells. So to say this another way, our cells need to constantly catabolize nutrients, burn glucose, burn some other fuel. They have to do this because they have to keep that ratio charged in a range where that can then support other cell functions, allow them to fight entropy and maintain order in biology. We alluded last time if you stop making ATP, well, what's going to happen? Well, that ATP-ADP ratio the cell has is going to fuel whatever reactions it can do until suddenly that ratio falls below the point where it's useful to drive those unfavorable reactions. In fact, if you stop ATP production, you will consume that ATP-ADP ratio in seconds. And this is why if you poison ATP production, cells die very quickly. Remember last time we mentioned you have a heart attack, blood flow stops going to a tissue. No more nutrients and oxygen. Cells die quickly. Cyanide poisoning does the same thing. Cells die quickly if you stop their ability to maintain this ATP-ADP ratio in the right range. And so remember the concentration, the absolute concentration of ATP for these reasons is not a good indicator of cell energy. The correct indicator of cell energy is the ratio of ATP to ADP. This term is sometimes referred to as the energy charge. And so that can be the ATP to ADP ratio, or oftentimes more correctly is the ATP to AMP ratio. Why the ATP to AMP ratio? Well, that's because once you understand this, you can now understand that cells have a failsafe mechanism to protect themselves if this ratio of ATP to ADP becomes too low. What is that mechanism? Well, you can carry out this reaction. Take two ATP molecules and turn them into transfer a phosphate to make an ATP plus an AMP. Why is that a failsafe mechanism? Well, if they care about this, if the ATP-ADP ratio is what maintains your ability to drive these thermodynamically unfavorable reactions, if I sacrifice the denominator by removing an ATP and get an ATP out of it, I've now at least temporarily fixed that ratio. Now this is something that obviously cannot go on forever.

~10:36But in the short term, it does allow cells to protect that ratio. And in fact, what cells actually defend is this ATPAMP ratio. And it's very, very difficult to change the ATP-AMP ratio in a cell and actually have it live. So it's really this ATP-AMP ratio that is most formally related to what would be called energy charge. But this is really why this is important is for all the reasons that I just described. All right. So hopefully, it's clear to you now why cells have to keep ATP-ADP ratio in the right range, why this is useful for them to now carry out all of these unfavorable reactions-- fight entropy, stave off death. And they can even have a failsafe here and sacrifice ADP as a way to try to maintain that as a very last resort. It also says why cells have to always do catabolism, because you always need the energy input to keep that ratio in the right range. However, you might imagine that, in real life, the messiness of biology, that there's lots of situations where the demand for ATP will suddenly increase. Well, of course, maybe you can just metabolize more sugar, do more nutrient catabolism to try to keep that ratio high. But that takes time. And so you need ways for cells to quickly respond to then allow them to mobilize all of this extra thing to-- extra fuel to do that. For example, we're walking around our ancestors on the plains somewhere and a lion appears. We don't have time to mobilize a bunch of new fuel. We have to be able to run away really quickly or the lion's going to eat us. And so cells have systems to suddenly respond to I need a lot more ATP demand to run away by having ways that one can buffer this ATP-ADP ratio, protect it in the short term, and allow cells time to ramp up nutrient metabolism in order to keep this high. All right. Well, if we wanted to create such a buffer system, how would we actually do it? Well, obviously, we have to follow all the rules of thermodynamics that we discussed. And we have to do so in a way that actually illustrates how it is that very well how metabolism works. In other words, if suddenly the ATP-ADP ratio drops, if we have a sudden drop in ATP-ADP ratio because we have increased demand, needing to run away from a lion, how could we actually create a buffer system that protects this? Well, to do it, we would need to couple ATP-ADP to some other product reactant pair, let's call them x and y, such that the ratio of y to x is coupled to the ratio of ATP to ADP. And you want it coupled in such a way that if that ratio goes down, this ratio can now protect ATP, basically favor ATP to ADP synthesis. However, when nutrients are plentiful and this ratio is really high, it then in turn can charge up this ratio so that it can buffer it later when the ATP-ADP ratio starts to fall. Now the most famous example of this actually comes from muscle physiology. And it's best described in terms of muscles and how they quickly respond to increased demand, like that running away from a lion. However, it in fact applies to many, many cells, including most non-muscle cells in animals.

~13:47All right. And I should say that it turns out that if you turn off ATP synthesis in a muscle, people have estimated that your ATP-ADP ratio will fall into a range where it is no longer useful to do anything within a matter of seconds, some claim even less than a second. And so this system ends up being very important.

~14:08Now the system that muscle uses, and many other cells do as well, is something called the creatine/creatine phosphate system. Now if you guys are weightlifters or bodybuilders out there, you probably have heard of creatinine because it's a very popular supplement that is taken for weightlifting, something I obviously don't do a lot of. But that creatine/creatine phosphate is out there. I'll describe to you what it actually does for muscle cells. And you can decide for yourself if this is actually useful as a energy or weightlifting supplement. So what is creatine/creatine phosphate? What does it look like? Well, it's basically looks like this. By the way, I will use this abbreviation often throughout this course, just a shorthand to draw a lot of these structures. So this here would be a carboxylic acid group drawn like this or like that, drawn to the carboxylic acid group just so you understand my shorthand. OK. So this here is the molecule creatine. If I put a phosphate here, this now becomes creatine phosphate. Again, just to remind you, this P with a circle over it is a phosphate group. I'll draw this particular phosphate group because it's a nitrogen-phosphate bond, a phosphoramidate bond. This is actually a bond that is more labile than the PO bonds that we're used to seeing in the phosphodiesters like ATP, et cetera. All right. So that's creatine and creatine phosphate. And if we draw out the reaction, creatine phosphate goes to creatine plus inorganic phosphate, so hydrolysis of creatine phosphate. This has a delta G0 prime of minus 10 kcals per mole. What does that mean? It's negative. Tells us about the equilibrium constant. Equilibrium lies to the right. Remember ATP to ADP hydrolysis shown over there, that hydrolysis has a delta G0 prime of minus 7.5 kcals per mole. Equilibrium also lies to the right. This number is more negative. This equilibrium lies further to the right than ATP hydrolysis does. So now what happens if we couple this reaction to the ATP hydrolysis reaction? So now we can do ATP plus creatine goes to creatine phosphate plus ADP. So we've coupled those two reactions together. So delta G0 prime, ATP to ADP is minus 7.5. Creatine phosphate to creatine in that direction is negative 10. In this direction, it would be positive 10. And so as I've drawn the reaction, this delta G0 prime will be plus 2.5 kcals per mole. 10 minus 7.5 plus 2.5, which tells us then that this equilibrium now lies to the left. However, which direction this reaction actually goes is not determined only by the equilibrium. It's really determined by delta G. So we can calculate that. So delta G equals delta G0 prime plus RT log of, as we've drawn it, creatine phosphate over creatine and ADP over ATP. So you can now imagine that this term here is the sum of those two. So it's plus 2.5. And so if this term is less than 2.5, what does that mean? Well, if it's-- I'm sorry. Less than negative 2.5, then delta G will be negative and you will favor creatine phosphate production. If this term is greater than negative 2.5, then delta G will be positive and you will favor ATP production. Why is that important? Well, if you think about the conditions, how you could make this term less than 2.5 or less than negative 2.5 or greater than negative 2.5, basically, what's going to happen is that if the ATP-ADP ratio is high, lots of energy around, no problems charging up, lots of things to use, charge up this ATP/ADP to a very high ratio, well, now you will favor this direction, creatine phosphate synthesis.

~17:33However, if ATP/ADP begins to fall and is lower, well, now this term is going to flip and you'll now favor ATP production. And so basically, by charging up this creatine/creatine phosphate ratio, it now creates a situation where it can buffer that ATP/ADP ratio so that when times are good, you charge up the buffer. When times are bad, you then consume the buffer in a way that allows you to protect your ATP/ADP ratio for a short time. And this is illustrated very nicely here in this slide that basically shows what happens in muscle physiology. And so this is just a theoretical curve that if you suddenly need to contract your muscle and you stop ATP production, well, ATP levels will fall. That ATP/ADP ratio more correctly will fall rapidly within the cell. Creatine phosphate will then now kick in to protect that ATP/ADP ratio as you can ramp up either anaerobic or aerobic metabolism to now buffer that ATP/ADP ratio and keep that muscle functioning. Now I want to make one point because oftentimes biochemistry textbooks will refer to things like ATP or creatine phosphate as, quote unquote, "high energy compounds." And of course, I guess they are high energy because they have these labile phosphate-nitrogen or phosphate-oxygen bonds. And because they're labile, hydrolysis is favorable. And to a certain extent, it's that hydrolysis that is providing energy. But remember it's not the metabolite or the bond in isolation that's really providing the energy. It's these ratios of ATP to ADP, creatine phosphate to creatine, that allow you to couple, for all the reasons we've spent now a lecture and a half discussing, two other reactions that really provides biological energy. All right. Now we're ready to discuss how it is that we can use glucose oxidation, nutrient catabolism, to keep ATP/ADP high, that is in a useful range, where it can allow cells to do otherwise thermodynamically unfavorable things. So how could we make this happen? Well, just like this example with creatine and creatine phosphate, if we want to use nutrient catabolism to do the same thing, we need to couple reactions of glucose oxidation, nutrient catabolism, that will allow us to synthesize ATP, ADP to ATP, and have that synthesis be favorable despite the high ATP/ADP ratio in cells. So let's think about it. So what is this? Nutrient catabolism. What did we talk about last time? Oxidizing glucose to carbon and CO2. So here's our reaction for that. So here's carbohydrate, glucose CH2O6 plus six O2 goes to six CO2 plus six H2O. That's the chemical formula for burning wood. So delta G0 prime for this is minus 686 kcals per mole. Quite a negative number. Equilibrium greatly favors the completely burned state, oxidized state, of that carbohydrate. And so you can imagine that this equilibrium is so favorable to the right, if we couple this reaction to ATP synthesis, you're clearly going to have plenty of energy there in order to maintain that ATP/ADP ratio in the right range to be compatible with cells using it to do other things. All right. So how do we actually do that? Well, if we do it, we have to follow the same rules of thermodynamics and do the same things that we did before. Basically, we have to do this reaction in a way that we can draw out a similar type of equation as we did for creatine and creatine phosphate. And at the highest level, that is how nutrient catabolism will allow us to keep ATP/ADP ratio high even as ATP hydrolysis is constantly fueling all these other unfavorable processes in the cell.

~20:55Now if we did this all in one step, our burning wood example, lots of heat is generated. That's too much energy. Not so good for biology. But rather what biology does, as we alluded to last time, is that because what we talked about in the beginning, that all of the things that we discussed are true for individual reactions or many, many reactions, basically, if we turn A into B and we do it in one step, like burning wood, or we do it in 500 steps, the thermodynamic consequences are exactly the same. And so what we need to do is do stepwise catabolism of glucose and take advantage of the fact that that delta G change is the same whether it's one step or many steps and in that stepwise fashion build in reactions where we create intermediate chemistries that allow us to do things like we did with creatine and creatine phosphate such that we can favor ATP synthesis when ATP/ADP ratio is in the physiological range. And in essence, this is the magic of how biology does this and basically couples nutrient catabolism in order to provide energy. And so the pathway that we're going to discuss today and in the next lecture is something called glycolysis, which simply put is glucose lysis. It's the stepwise chemical oxidation of glucose, which we know from our burning wood example is favorable. And we're going to use this pathway that basically will be a pathway to allow the stepwise lysis of glucose, a favorable reaction, to individual steps that set it up in a way such that ADP to ATP synthesis is favored despite a physiological ATP/ADP ratio. Now I want to point out that this really illustrates, back to that high energy compound thing, that ATP is not the only energy currency in the cell. In fact, glucose is much more energy for a cell than ATP. You get much more energy released from burning wood than you get from hydrolyzing ATP. And in essence, this is why we use glucose-- why nature has used glucose, and other molecules like it, as an energy storage device, because here is a relatively stable molecule, can sit around for a long time, and you can burn it when you need it to keep your ATP-ADP ratio high. All right. So, at the level of reactions and pathways now, how can we use glucose oxidation and couple it to drive ATP synthesis? Well, hopefully it's very clear that we have to basically set up a situation like this creatinecreatine-phosphate system that we already discussed. And so I want to start by discussing, what are the reactions in glycolysis that ultimately behave like this and allow this favorable ADP-to-ATP synthesis? And then we will go ahead and consider how we can make those reactions work in the context of an entire pathway that allows glucose oxidation. All right. Now, like creatine, phosphate, and creatine, both of the steps in glycolysis that favor ATP synthesis involve intermediates where you have phosphate on some molecule, plus ADP goes to ATP plus the phosphate coming off that molecule. Now, if we go to our creatine-creatine-phosphate system, obviously this equilibrium needs to lie to the right. And so the first one of these is as follows. It involves this molecule, phosphoenolpyruvate, also referred to as PEP. You will see throughout the course that we love to give these little acronyms to our metabolites because it's hard to constantly say and write out phosphoenolpyruvate. Much easier to say PEP. So phosphoenolpyruvate plus ADP goes to ATP. Plus, if I take the phosphate off of there, I get this molecule. This molecule is enolpyruvate. Enolpyruvate can be rearranged as follows. To make this molecule a ketone pyruvate.

~24:24You'll note-- hopefully remember from your organic chemistry classes that ketones like pyruvate can exist in two forms. They can exist in the ketone form or the enol form, rearranging via the chemistry that I've shown. The keto form is greatly favored over the enol form. However, by having a phosphate trap pyruvate the enol form-phosphoenolpyruvate, when you remove the phosphate and it's wanting to exist in the keto form, greatly enhances the energetics of this-- basically makes the reaction, the equilibrium want to lie very far to the right. And by the way of chemical tricks like this and others like it, you will see used over and over in biology. In fact, this is illustrated, if we asked what's delta G0 prime for this reaction, it's minus 15 kcals per mole. That tells us that the equilibrium of this reaction lies to the right. In fact, it lies far to the right, and in fact, this is-- you may remember ATP hydrolysis is negative 7.5 kcals per mole. So lies twice as far to the right as ATP hydrolysis does. And in fact, this minus 15 kcals per mole is the most favorable, at least in terms of individual reactions that at least I know of in metabolism. All right. Well, if this equilibrium for this is PEP to pyruvate, this here is delta G0 prime for ATP-ADP. So if we want to know what's delta G0 prime for the entire reaction, well, if PEP to pyruvate is minus 15, ATP to ADP is minus 7.5. Well here, we're going in the opposite direction, so it'd be plus 7.5. So it's minus 15 plus 7.5, means that for the entire reaction, this is minus 7.5 kcals per mole.

~26:00Means that this is favorable, equilibrium lies to the right. If we drew out-- I'm not going to do it again because I've done it a lot, but if we redrew out this equation, replacing pyruvate and PEP in the correct places, there for creatine, phosphate, and creatine, you would see that there's lots of conditions within cells that will favor ATP synthesis even when ATP-ADP ratio is high. All right. This reaction is catalyzed by an enzyme called pyruvate kinase. And pyruvate kinase is a classic example of a, quote-unquote, "irreversible reaction." However, I want to point out that the enzyme pyruvate kinase is actually named for the reverse reaction that's supposedly irreversible. Pyruvate kinase. If you take pyruvate and you act on the kinase as you learn from Professor Yaffe, that phosphorylates something. You phosphorylate pyruvate, you get phosphoenolpyruvate pyruvate kinase. And so remember that absolutely no reaction is reversible. It's only reversed-- what they mean by irreversible when they say pyruvate kinase step in glycolysis is irreversible, is that it is effectively irreversible under the conditions that are compatible with life that exist in cells. Of course, if you have very low ADP and very high ATP, you can come up with a way to net synthesize PEP. You just can't drive the reverse reaction under the ATP-ADP and the PEP pyruvate ratios that physiologically exist in cells. However, this is set up in a way such that you can favor ADP-to-ATP synthesis by converting PEP to pyruvate in a pathway, as we will see happens in glycolysis. All right. Now, the second reaction that I want to-- that allows ATP synthesis in glycolysis is catalyzed by an enzyme called phosphoglycerate kinase. And this carries out the following reaction. OK. So this molecule is 1,3-Bisphosphoglycerate, abbreviated 1,3-BPG. And basically the phosphate is transferred from here to ADP, which gives you this molecule, 3-phosphoglycerate or 3-PG.

~27:44So this reaction, the delta G0 prime for the entire coupled reaction, both steps of the reaction, is minus 4.5 kcals per mole. Tells you about the equilibrium constant. Equilibrium lies to the right, doesn't lie as far to the right as the PEP plus ADP goes to ATP plus pyruvate reaction, but nonetheless, is one where there's lots of conditions that are favorable in cells to maintain a high ATP-ADP ratio, although we will see later that this reaction actually runs quite close to equilibrium in cells, so there's actually conditions where it can go in the opposite direction as well, and we'll get to that later in the course. I want to point out one thing about this, one thing that makes this reaction favorable, as you notice here that this is a phosphate on an acid group, a so-called acid anhydride. And this is a pretty good phosphate donor. And so you want to lose that phosphate, which is one of the reasons why this reaction is favorable for ATP synthesis even at high ATP-ADP ratios. All right. So these are the two reactions in glycolysis that can favor ATP synthesis at a high ATP-ADP ratio just like creatine and creatine phosphate. And so hopefully now it's become apparent to you that if during our pathway of glucose catabolism we can build a pathway that couples what we already know is favorable, glucose oxidation, to generate these intermediates, PEP and 1,3-Bisphosphoglycerate, lacerate we can then build a pathway where those reactions can make synthesis of ATP favorable even though there's a high ATP-ADP ratio in cells, and in fact, maintain that high ATP-ADP ratio in cells. And in essence, this is the logic or the goal, if you will, of glycolysis and why it's important to provide energy to cells. OK. All right. Now, if we're going to make a pathway where glucose oxidation is coupled to making PEP and 1,3Bisphosphoglycerate, it's obvious we need to add phosphate to the system. There's no phosphate molecule on glucose. Now already mentioned earlier-- and I just erased it-- that to trap glucose in the cell, the first step in glycolysis, you add phosphate. But that phosphate donor is ATP. And so ATP being the phosphate donor doesn't help us here. If we're going to use phosphate transfer here, we need to have phosphate added into the system if this is going to work. We can't have ATP as our phosphate donor. You also saw lots of reactions from Professor Yaffe that involved phosphate transfer reactions. But again, ATP-- or GTP is the phosphate donor. And so for this system to work, we need a way to get inorganic phosphate onto a carbohydrate or we can't make things like PEP or 1,3-Bisphosphoglycerate that will allow ATP synthesis. Now we already saw earlier that additional phosphate to glucose is not favored. Remember, delta G0 prime of phosphorylating glucose was plus 3.3 kcals per mole. And it turns out that says equilibrium lies to the wrong side, so we can't net do that without ATP or energy input. And it turns out this is true for other sugars as well. And so you need energy input to add phosphate to the sugars. Plenty of energy input from glucose oxidation, but we need a reaction where phosphate addition is coupled to another reaction that makes it favorable, and we have to do this without making ATP. And again, it illustrates, again, the biological energy is not just about ATP. At a deeper level It's something different. And so to really discuss this and understand it, I need to introduce two concepts that are going to come up again over and over again in this class and across metabolism. The first of these is a very high-level bioenergetic concept, although it's really an energetic concept, and it comes back to what we already talked about.

~31:19So, we know burning wood is favorable. The CN H2O N plus oxygen going to CO2 plus water. That's burning wood, lots of energy released. Drew it up there for a single-glucose molecule and what the energy release the delta G0 prime is for that reaction. Now I want to point out chemically what is going on in this reaction. Chemically what is happening for this to occur is that electrons from the carbon are being moved to the oxygen. You are changing the oxidation state of carbon and of oxygen in order to carry out this reaction. Oxygen is a better electron acceptor than carbon, and so transferring those electrons from the carbon to the oxygen is favorable. That is, in essence, what is going on in this very favorable reaction to burn wood. Let me illustrate that a bit more explicitly here. So the carbon-- most of the carbons in carbohydrates are alcohols. So here, you can call this our carbohydrate, with the exception of the ketone or the aldehyde if it's a ketose or an aldose. All the other carbons in the sugar are alcohols. All the other bonds there. And so if we oxidize that carbon-- that is, if we remove electrons from the carbon, how can we do that? So this here generates this H- hydride ion. This is, in essence, two electrons that we are removing from the carbon. That gives us a ketone. So if we oxidize the alcohol and we remove two electrons, we get the ketone. We could oxidize this ketone further. That will give us the carboxylic acid. If we oxidize it even further, now we get CO2. So, moving in this direction, this is oxidation. Transferring electrons from the carbon to the oxygen to go in this oxidation direction, that is glucose oxidation is what's happening when we're burning wood. It's what happens when we burn glucose. Energy is released. This is largely what catabolism is about in biological systems. Now then reasons, then, that if we're going to move in the opposite direction, so-called reduction, that is going in this direction. Reduction. That's largely energy storage or anabolism in metabolism. We are storing energy that we can burn later. And of course, what's the more extreme version of this? Well, what if we add two more electrons to the carbon going in this direction? Well, what do we end up with? Well, now we end up with the saturated carbon. more reduced than the alcohol is the fully saturated carbon. You will see later that this is what fat is. You saw that in some of the earlier lectures. It's also what gasoline is. What's gasoline? It's fully saturated hydrocarbons that we then burn to CO2 and derive energy. Just like burning wood, burning gasoline all in one step, oxidizing the gasoline gives you energy. There's more energy in gasoline. You can release more energy per weight from gasoline, from fully-saturated hydrocarbons than you can from wood. Same thing happening here. What has more calories in your food? Everyone knows fat has more calories per weight than sugar. It's because the more reduced our carbon is, the more energy it stores, and as we oxidize that carbon, that is what releases the energy. And so energy transduction in biological systems-- and actually, in non-biological systems, as I just illustrated with the wood and gasoline example, but definitely in biological systems, is largely about oxidation and reduction reactions, and you are going to see this coming up over and over again.

~34:34Now, based on what I just said, the real magic, if you will, of glycolysis can be further refined to say that what glycolysis does is it couples the favorable oxidation of carbon with reactions that allow phosphate additions and production of intermediates, like PEP and 1,3-Bisphosphoglycerate, where synthesis of ATP is favored despite the high ATP-ADP ratio in cells. If you think about it, that process then allows you to keep that ATP-ADP ratio in the range where it can then be useful for all the reasons we've been talking about now for two lectures. Allows the cell to couple that ATP-ADP ratio, that-- in ATP hydrolysis to otherwise carry out otherwise unfavorable reactions. OK. So, based on that, arguably the key, or at least a very important reaction in glycolysis, is the oxidation step which happens to add the phosphate and also happens to make 1,3-Bisphosphoglycerate, which we saw earlier can be used to drive ATP synthesis. And so that reaction is the following. So this molecule hopefully looks familiar to you from the carbohydrate lectures. This is D-glyceraldehyde 3phosphate. I will abbreviate it frequently is G-3P for glyceraldehyde 3-phosphate, but it's D-glyceraldehyde 3phosphate. Remember, it's a D, sugar, because the OH group is pointing to the right as we drew it this way. It's also an aldose because it's a aldehyde sugar, and it's phosphorylated on the 3 position-- remember the way we numbered sugars was starting at the carbonyl-- 1, 2, 3. This is carbon-3, phosphorylation on the 3 position. Glyceraldehyde 3-phosphate. OK. So, this step generates 1,3-Bisphosphoglycerate. Well, to do this, there's actually two things that happened here. First we had to add the phosphate to this. It says two phosphates on it. bisphosphoglycerate, this has a single phosphate. So inorganic phosphate has to be added to generate that molecule. By the way, adding that phosphate delta G0 prime is positive plus 1.5 kcals per mole. That means even equilibrium, this reaction would lie to the left, of course, because delta G0 prime is positive. However, remember the next step downstream of this is the one shown over here where we do ATP synthesis. That step is very favorable. So this is an example where metabolism has coupled a very favorable step to a step that's less favorable. That can keep the 1,3-BPG levels low and pull the entire system forward, the metabolic trick we discussed in the last lecture. OK. You'll note the other thing that's happening here, other than adding that phosphate group, is that we've changed this aldehyde to a carboxylic acid. That is an oxidation reaction. Ketone to the acid is an oxidation reaction. I will illustrate over here this so you can see why it's an oxidation reaction. So here's our aldehyde. If we take electrons from the carbon and that generates this hydride ion to electrons, what are we left with? Well, we're left with this intermediate that will never exist with a positive charge on the carbon. Water, which, of course, is abundant in biological systems. And then come-- OK. And it basically illustrates that as I go from the aldehyde to the acid, of course, I make a proton and a hydride ion, but really, I have changed the oxidation state of the carbon from the ketone to the acid state. Two electrons are lost there, is shown as that hydride ion. OK. So, what does that mean? Well, that means that we have to maintain that balance in this reaction as well. So water is going to have to come in and a proton will be produced. But the key thing is we also generate two electrons, this hydride ion that have to go somewhere. Now, finding a place for those two electrons to go is what brings up the second high-level concept that I now need to spend some time discussing.

~38:08And this second high-level concept that I need to introduce is the role of cofactors, and cofactors also is effectively the role of vitamins and what role that these things play in metabolism. Most vitamins are used to support metabolic reactions, and we're going to learn all about what many of the vitamins that you guys are familiar with reading the side of your cereal box actually do throughout the course.

~38:31Now cofactors, as I said, often involve vitamins, are molecules that provide useful chemical groups that facilitate the reaction chemistries that are needed in metabolism. So they provide some useful chemical groups to facilitate some of the chemistries that we need to carry out metabolic reactions. They are, in general, in small non-stoichiometric quantities in order to carry out this function. What do I mean by that? Meaning that you can imagine that the cell will convert lots of glyceraldehyde 3-phosphate to 1,3Bisphosphoglycerate, but the enzyme, you don't need one enzyme for every time you convert that reaction. The same enzyme can do it over and over and over again. Well, it turns out, cofactors are much more like enzymes in that respect. They need to be recycled in a way that they can catalyze the turnover of reaction many, many, many times, and it'll be much clearer what I mean by this when we get into some of the details. Now, the vitamin concept comes from the fact that often these cofactors or parts of the cofactors are not made by animals, not made by humans. If they're not made by us, but we need them to do our metabolism, well now we have to get them from the diet, and this is really the concept of vitamins. These are small things that we have to get in quantities from the diet, and it turns out they're very important to help us carry out the metabolism we do. As an aside, the vitamin industry is a very big business. Lots of unfounded claims out there. I'm not going to get into-- that can get very political. I'm just going to say that just know that more is not always better, and the goal for this class is really to understand what some of these vitamins actually do. OK. Now, the relevant cofactor for the reaction that we're talking about is a cofactor called nicotinamide adenine dinucleotide, which is always abbreviated NAD or NAD+. This stands for nicotinamide adenine dinucleotide. You see why we want to abbreviate that NAD. And basically what nicotinamide adenine dinucleotide is is it's a factor that can solve our problem of where to put these electrons in this oxidation reaction. That is, you can accept two electrons from a reaction like this one here and cycle to another form of the cofactor called NADH. All right. What is nicotinamide adenine dinucleotide? Well first, let's draw what it looks like. OK. Sorry it's squeeze down here a little bit. So this here is adenine. This group up here is nicotinamide. So if you look at this, this is a dinucleotide. And so down here, this, of course, is, from here down, is AMP. That phosphate is now over here. This is a separate nucleotide, where rather than having one of the AGCTU bases that you're used to from RNA and DNA, nicotinamide is the base. And so it's this AMP plus this nicotinamide base is a monophosphate, or a nicotinamide adenine dinucleotide. This, the nicotinamide group, is derived from a vitamin called niacin. And so from the side of your cereal box, this is what niacin does. It allows you to generate the nicotinamide group that's part of this cofactor, nicotinamide adenine dinucleotide. Now, nicotinamide adenine dinucleotide is useful because of what that nicotinamide group can do.

~41:36And so I'm not going to draw out the whole molecule, but I'll show you. So NAD+, which is the oxidized form of nicotinamide adenine dinucleotide, is as I drew it up there. So this here would be attached to the rest of the dinucleotide. So this is the oxidized form. I can have-- here's my hydride ion, which, remember, is two electrons. If I carry out that chemistry to add those two electrons to the nicotinamide group, you now end up with the reduced form of nicotinamide adenine dinucleotide NADH. So the difference between NAD+ and NADH, it's the H, but it's really the two electrons that were added in adding that H. So it's oxidized form and reduced form. So remember, I want to stress this again, at least in biology we can't create or destroy matter. You can do that in nuclear physics, but we can't do that in biology. And so that means that if we do an electron transfer like we did in this reaction where we oxidized the aldehyde in glyceraldehyde 3-phosphate to the acid in 1,3Bisphosphoglycerate, those electrons have to be accounted for. And so if one molecule is oxidized, another one has to be reduced. So redox reactions always-- that is, redox, oxidation-reduction reactions, always have to happen in pairs. And so over there we have the aldehyde in glyceraldehyde 3-phosphate is oxidized to form the acid in 1,3-Bisphosphoglycerate. As part of that reaction, we can couple it such that the nicotinamide group, which is oxidized in NAD, can be reduced to the nicotinamide group that is found in NADH. So that means we can draw this in here, is that having that cofactor coupled to this reaction now allows us to fix the redox problem of this reaction. Now this reaction here is catalyzed by an enzyme called glycerol 3-phosphate dehydrogenase. I'll draw that out in the second. Often abbreviated GAPDH, the loading control in all of your western blots, is the enzyme GAPDH, glyceraldehyde phosphate dehydrogenase. That is the enzyme that carries out that reaction. Now notice, the name fits. It's a dehydrogenase, it's removing a hydrogen, removing two electrons as a hydride ion, but it's really-- what's important here is the electrons moving, not the hydrogen itself. So in that way it's a little bit misleading. And you will see that in general, dehydrogenases are classes of enzymes that catalyze oxidation-reduction reactions, and as a result, often involve NAD/NADH as cofactors because they facilitate those electron transfer reactions. OK. So now we're able to describe how GAPDH works in great detail. And so the chemistry of GAPDH works as follows. And so in the active site of GAPDH, there's a cystine residue. That cystine residue has a sulfur shown here. Also bound near the active site is in NAD molecule to catalyze the reaction shown. That NAD has to be in the oxidized state, so it's NAD+. And so glyceraldehyde 3-phosphate binds in the active site. There's glyceraldehyde 3-phosphate. By the way, I will show you how many reactions in metabolism work chemically. Obviously we don't have time in this class, an overview class to go into all the great chemical details of what's going on and exactly how everything works, but I'll at least try to give you a flavor of some of the chemistry that happens throughout the course. All right. So once you have, then-- OK. So now you have this intermediate bound into the enzyme active site. You can now carry out the oxidation of that carbon. So these are two electrons being removed as a hydride ion. They can be transferred to NAD in the active site. That generates NADH by the exact chemistry that I just showed you. OK.

~45:03That NADH now needs to be exchanged out or the enzyme will not be able to catalyze the next series of reactions, and that NADH will then go somewhere else, and of course, has to be cycled back to NAD, but that doesn't happen in this reaction, per se. OK. Next, this here, you'll notice, is that by the way this chemistry worked, we formed this thioester bond in the active site. This is another thing that you will see over and over again in metabolism. This sulfur in the thioester bond is a good leaving group, and so it's really setting up a situation that will now help with the phosphate addition. So here, I'll draw out inorganic phosphate. And that, of course, will give us the identical enzyme active site that we started with plus 1,3-Bisphosphoglycerate. And that 1,3-Bisphosphoglycerate can now be coupled to ATP synthesis to make 3-phosphoglycerate. And so you can see, in essence, what this reaction has done is that it has coupled oxidation of the aldehyde to the acid, the aldehyde in 3-phosphate to the acid in 1,3-Bisphosphoglycerate, which can then do phosphorylation of ADP to make ATP. So in essence, this is oxidative phosphorylation. Now I realize in high school or in other classes, oxidative phosphorylation means something different to you, and of course, we will discuss the process that is called oxidative phosphorylation in great detail later in this course. But I want to point out that the chemistry that's actually happening here in glycolysis is also oxidative phosphorylation. It's basically how you coupled this favorable carbon oxidation that's occurring at this GAPDH step in a way that allows you to synthesize ATP at a high ATP-ADP ratio, which in the end will allow cells to do otherwise unfavorable things. OK. Now we're ready to discuss these reactions, the GAPDH reaction, the pyruvate kinase reaction, the phosphoglycerate kinase reaction, and how they fit into glycolysis as a metabolic pathway in a way that allows cells to use glucose metabolism-- that is, the breakdown of glucose, the burning of glucose as a way to get energy. And that pathway is, of course, glycolysis or glucose lysis that we mentioned earlier. Glycolysis is a very ancient and ubiquitous pathway. It's used by essentially all life on Earth and probably has been for at least 2 to 3 billion years. Glycolysis, I guess, as a pathway was, quote-unquote, "discovered" mid-19th century by Louis Pasteur, and Louis Pasteur, of course, described life, microorganisms as being responsible for the process of fermentation. That is, conversion of glucose into alcohol, something many college students are interested in, as well as other organic acids. And of course, fermentation to make alcohol or to store other types of organic acids has been used in food preservation for centuries, which is really why Pasteur's discoveries were so impactful at the time. Now even though Pasteur discovered this, he didn't really understand chemically how it worked, and it actually took decades to figure that out. And when it was finally figured out and described as the pathway called glycolysis, this was done by two chemists, two chemists in Germany, Embden and Meyerhof. And basically what they did is they pieced together several enzymatic or chemical activities that have been purified from cell lysates mostly from yeast and used those to build a pathway that described chemically how you could start with glucose and chemically break down that glucose in a way that made sense in this fermentation pathway, and that is why glycolysis is sometimes called the Embden-Meyerhof pathway.

~48:20Now, glycolysis does not completely convert glucose to CO2. Instead, what glycolysis does is it converts glucose-so I will draw here in the pyranose form. It converts glucose by a number of steps into two molecules of the organic acid pyruvate, which, of course, was the product of that pyruvate kinase reaction that we showed earlier. Note that the fermentation that Louis Pasteur described was further metabolism of that pyruvate into something else. In the case of yeast, it was ethanol or ethyl alcohol, or something else. The fermentative product in mammals is this organic acid lactate. And what we will see next time is that this fermentation of pyruvate into one of these other molecules ends up being an alternative to the further oxidation of pyruvate to CO2 which requires oxygen, and the reason oxidizing pyruvate to CO2 requires oxygen is because ultimately we have to regenerate that NADH we made in glycolysis to allow that cofactor to cycle and glycolysis to keep moving. Fermentation gives us an alternative way to do this, which we'll discuss in great detail in the next lecture. But first I want to focus on glycolysis and what we can describe next time as really how we can build a pathway to turn glucose into pyruvate. This glycolysis, this Embden-Meyerhof pathway, and some of the details of that pathway are shown here on this slide. And this is where we'll start off the next lecture, discussing how each of these steps in glycolysis function in a way that allow a pathway to generate phosphoenolpyruvate, 1,3-Bisphosphoglycerate, incorporate this GAPDH reaction, and do so in a way that's overall energetically favorable to allow the cell to couple this oxidation of glucose to keeping ATP-ADP ratio in the right range to support other unfavorable functions in the cell. OK, thanks.

1.6ATP 能量通货、磷酰基转移势能阶梯、高能硫酯键与能荷调控：为什么我们早餐不直接吃 ATP？

一、灵魂拷问：为什么“ATP 绝对浓度 = 能量”是大错特错？为什么我们早餐吃麦片而不吃纯 ATP？

在 L20 课堂上，Vander Heiden 教授写下己糖激酶的实际自由能方程后，用粉笔重重圈出了分母上的 [ATP] 和分子上的 [ADP]：

Δ G = Δ G'^° + RTln≤ft(([G-6-P])/([Glc]) · ([ADP])/([ATP]))

他严肃地对全班说：“请大家千万不要犯无数生物学家常犯的低级错误——以为‘ATP 就是能量，细胞里只要有很多 ATP 就有很多能量’！看清楚这个对数项：如果我把细胞里的 [ATP] 提高到天上去，但与此同时 [ADP] 也一样高（即 [ATP]/[ADP] 处于平衡比值），那么 ATP 所能提供的驱动力是 零（Zero）！反过来，即便细胞里 ATP 的绝对浓度很低，只要 [ADP] 比它还要低得多（即维持极高的 [ATP]/[ADP] 比值），ATP 依然拥有强大的做功驱动力！”

这一下子就彻底回答了章首的灵魂追问：为什么我们早餐吃富含淀粉的麦片/土豆，而从来不直接吃 ATP，细胞也不能把 ATP 当作长期储能物质？

1. 对数规律的指数级耗能诅咒：因为 Δ G 与 ln([ATP]/[ADP]) 成对数关系！如果你想通过单纯把 [ATP]/[ADP] 比值再推高 10 倍、100 倍来“多存一点能量”，你需要投入的合成能量将呈指数级暴增，很快就超出了营养氧化所能提供的自由能极限；反之，一旦停止产 ATP，只要消耗掉一丁点 ATP 变成 ADP，分母 [ADP] 从极小值翻几倍，[ATP]/[ADP] 比值就会在短短 1～2 秒内断崖式暴跌，瞬间跌破驱动吸能反应所需的阈值！
2. 分子质量与跨膜障碍：ATP 分子量高达 507 Da，且带有 4 个负电荷，既不能穿过肠上皮细胞膜（吃进肚子里也会在肠道被水解成核苷和磷酸），而且储存 1 摩尔可转移磷酸基团居然要背负 507 g 沉重的核苷酸骨架！因此，ATP 绝不是细胞的“长期能量储库（Storage）”，而是随赚随花、每秒钟都在高速周转循环的“流动现金通货（Energy currency）”；真正的长期能量储库是化学性质稳定、能量密度极高的还原态碳聚合物——多糖（淀粉、糖原）和三酰甘油（脂肪）！

二、ATP 的结构、水解释能三大原因与“基团转移”真相

让我们回到王镜岩教材第 16 章，拆解腺苷三磷酸（ATP）的分子结构与热力学参数：

ATP 由腺嘌呤（Adenine）、β-D-呋喃核糖和三个依次相连的磷酸基团（由内向外分别标记为 α、β、γ 磷酸基）组成。其中，α-磷酸与核糖 C5'-OH 之间是普通的磷酸酯键（Phosphoester bond），水解仅释放约 -14.2 kJ/mol 自由能（低能键）；而 α 与 β、β 与 γ 磷酸基之间则是两个酸酐键——焦磷酸键（Phosphoanhydride bonds），习惯上用 Lipmann 引入的波浪号 ~P 表示“高能磷酸键”：

• 水解脱去末端 γ-磷酸（生成 ADP + P_i）：Δ G'^° = -30.5 kJ/mol（-7.3～ -7.5 kcal/mol）；在活细胞生理浓度下，实际 Δ G_p（磷酸化势）高达 -50～ -65 kJ/mol！
• 水解脱去 β,γ-焦磷酸基（生成 AMP + PP_i）：Δ G'^° = -45.6 kJ/mol（或计为 -32.2 kJ/mol，随后 PP_i 水解再放能 -33.5 kJ/mol）。

三、四大类高能化合物、磷酰基转移势能阶梯与高能硫酯键

在生物体内，高能化合物绝非只有 ATP 一种。按高能键的化学本质，王镜岩教材将其严格划分为四大类型（填空与选择题高频考点！）：

3D3D 结构库：ATP 与高能磷酸化合物构象查看 ATP 三磷酸静电排斥与 Mg²⁺ 螯合三维构象↗

结合上图（bioenergetics_coupling）与 Vander Heiden 教授在 L20 课堂上的精彩板书，我们必须深挖四个直击灵魂的原理问题：

1. 为什么 PEP（磷酸烯醇式丙酮酸，-61.9 kJ/mol）稳坐生物界“放能之王”宝座？

在 L20 课堂上，Vander Heiden 教授画出了 PEP 的结构：丙酮酸本是一个 α-酮酸，在水溶液中可以在酮式（Keto form）和烯醇式（Enol form）之间互变异构，但酮式比烯醇式稳定得多（酮式 ⇌ 烯醇式的平衡远远偏向酮式，自由能差高达约 -46 kJ/mol！）。

在 PEP 分子中，一个磷酸基团强行加在了烯醇羟基上，就像一把锁一样把丙酮酸死死困在了极不稳定的高能烯醇式构象（Trapped in the unstable enol form）中！一旦磷酸基团被转移给 ADP（脱去磷酸僅放能约 -15 kJ/mol），重获自由的烯醇式丙酮酸（Enolpyruvate）会在毫秒之内自发发生酮-烯醇互变异构（Tautomerization），瞬间翻转为极其稳定的酮式丙酮酸（Ketopyruvate），额外释放出高达 -46 kJ/mol 的巨大自由能！两项叠加，造就了 PEP 高达 -61.9 kJ/mol（课堂板书约 -15 kcal/mol）的惊人磷酰基转移势能！因此，丙酮酸激酶（Pyruvate kinase）催化 PEP + ADP → 丙酮酸 + ATP 的偶联总 Δ G'^° = -61.9 + 30.5 = -31.4 kJ/mol（板书 -7.5 kcal/mol），成为糖酵解末尾最强劲的产能引擎！（注意 Vander Heiden 教授的课堂幽默提醒：为什么这步生理上不可逆的产 ATP 酶偏偏叫“丙酮酸激酶”？因为早期生化学家是按它的逆反应——用 ATP 磷酸化丙酮酸来给它命名的！激酶（Kinase）：催化磷酸基团从 ATP（或其他高能磷酸化合物、核苷三磷酸）转移到底物受体分子（或反向从高能底物转移给 ADP）上的磷酸转移酶类，广泛参与能量代谢、底物水平磷酸化与细胞信号转导共价修饰 2021/2022/2023·名词）。

2. 为什么 1,3-BPG（-49.3 kJ/mol）是高能化合物，而 G-6-P 和 F-1,6-BP 却不是？

G-6-P、F-6-P、F-1,6-BP 和 3-磷酸甘油酸（3-PG）上的磷酸基，全部连接在普通的醇羟基（-CH_2OH）上，属于普通磷酸单酯（Phosphoesters），水解产物只是普通醇，没有额外的共振稳定或互变异构红利，故水解 Δ G'^° 仅有 -13.8～ -17.6 kJ/mol，统统不属于高能化合物 2008·单选！

相反，1,3-二磷酸甘油酸（1,3-BPG） 的 C1 位 是羧基（-COOH）与磷酸缩合而成的混合酸酐——酰基磷酸（Acyl phosphate）！反应物中羰基与磷酰基争夺桥氧电子且存在强烈电荷排斥；一旦把 C1 磷酸转移给 ADP，生成的 3-磷酸甘油酸（3-PG） 的 C1 羧基在 pH 7.0 下立即电离为具有高度对称共振稳定性的羧酸根离子（-COO^-），从而释放高达 -49.3 kJ/mol 的自由能，在磷酸甘油酸激酶（PGK）催化下轻松驱动 ADP 合成 ATP（偶联总 Δ G'^° = -49.3 + 30.5 = -18.8 kJ/mol）！

3. 为什么 ATP 的 Δ G'^°（-30.5 kJ/mol）不排在第一名，而是稳居势能阶梯的“正中间”？

请看图 bioenergetics_coupling 左侧的磷酰基转移势能阶梯：

如果 ATP 的水解自由能排在最高处（比如 -65 kJ/mol），那么分解代谢中的任何中间物都无法把磷酸基团转给 ADP 来给 ATP 充电；反过来，如果 ATP 的水解自由能排在最低处（比如 -10 kJ/mol），ATP 就没有足够的能量去给葡萄糖、果糖-6-磷酸或甘油加上磷酸！正因为 ATP 的磷酰基转移势能（30.5 kJ/mol）恰好处在势能阶梯的正中间，它才能完美胜任“通用能量中介（Universal intermediate carrier）”——向上可以接受超高能磷酸化合物（PEP、1,3-BPG、磷酸肌酸）转移来的磷酰基使 ADP 再生成 ATP（充电），向下又可以把磷酰基转移给葡萄糖、甘油等受体生成低能磷酸酯（放电做功）！

4. 当我们的祖先在非洲草原上突然撞见狮子：磷酸肌酸（Creatine phosphate）如何充当“ATP 缓冲池”？

Vander Heiden 教授在 L20 课堂上讲了一个生动的进化场景：当原始人在草原上散步突然撞见一头狮子，他必须在 0.1 秒内拔腿狂奔！然而，骨骼肌细胞内现成的 ATP 存量极少，一旦剧烈收缩，肌细胞内的 [ATP]/[ADP] 比值在不到 1 秒钟内就会消耗殆尽，而此时启动糖原分解和有氧呼吸还来不及！怎么保命？

骨骼肌和脑细胞进化出了一套基于质量作用定律的精妙磷酸原（Phosphagen）缓冲系统——磷酸肌酸 / 肌酸激酶系统：

磷酸肌酸（CrP） + ADP + H⁺ ⇌(肌酸激酶（Creatine kinase, CK）) 肌酸（Cr） + ATP

让我们跟着 Vander Heiden 教授算一算这步反应的自由能账单：

• 磷酸肌酸含有一个极不稳定的高能氮磷键（N～P，Phosphoramidate bond），其水解 Δ G'^° = -43.0 kJ/mol（板书约 -10 kcal/mol），比 ATP 水解（-30.5 kJ/mol，板书 -7.5 kcal/mol）更负！
• 因此，在偶联反应中：
  Δ G = Δ G'^° + RTln([Cr][ATP])/([CrP][ADP]) （顺向产 ATP 时 Δ G'^° = -43.0 + 30.5 = -12.5 kJ/mol，逆向储 CrP 时 +12.5 kJ/mol）
• 平时安静休息、营养充足时（When times are good）：线粒体氧化磷酸化源源不断产生 ATP，细胞内 [ATP]/[ADP] 比值极高，使得对数项扭转了 +12.5 kJ/mol 的平衡阻力，推动反应逆向进行（ATP + Cr → CrP + ADP），将多余的能量充电储存为高浓度的磷酸肌酸（肌细胞内 CrP 浓度是 ATP 的 3～8 倍，是肌肉收缩所需大部分能量的直接贮存形式） 2008·单选！
• 撞见狮子拔腿狂奔的最初几秒内（When times are bad）：肌球蛋白疯狂消耗 ATP 生成 ADP，只要 [ATP]/[ADP] 稍一试图下降，高浓度的磷酸肌酸立刻在肌酸激酶催化下顺向转移磷酰基给 ADP，瞬间把 [ATP]/[ADP] 比值死死托住（维持 4～6 秒极限冲刺），为糖酵解和有氧氧化的全面启动争取到宝贵的救命时间！（临床上心肌梗死时心肌细胞破裂，血液中肌酸激酶同工酶 CK-MB 急剧升高，也是诊断心梗的重要生化指标）。

5. 为什么硫酯键（如乙酰-CoA）没有磷酸基，却拥有媲美 ATP 的高能（-31.4 kJ/mol）？

在 L20 后半段讲到 3-磷酸甘油醛脱氢酶（GAPDH）催化机制及乙酰-CoA 时，Vander Heiden 教授反复强调了高能硫酯键（Thioester bond，R-CO～S-CoA 或 R-CO～S-Cys）在整个新陈代谢中的核心地位。

为什么普通的氧酯键（R-CO-O-R'）水解只放能约 -19 kJ/mol（低能键），而把氧原子换成同族的硫原子变成硫酯键（R-CO～S-R'），水解自由能就飙升到 -31.4 kJ/mol（与 ATP 水解的 -30.5 kJ/mol 旗鼓相当）？

因为在普通羧酸氧酯（R-CO-O-R'）中，氧原子的 2p 轨道与羰基碳的 2p 轨道大小高度匹配，氧孤对电子可充分离域发生共轭共振稳定（Resonance stabilization）；而在硫酯（R-CO～S-R'）中，硫原子属于第三周期元素，其庞大的 3p 轨道与羰基碳较小的 2p 轨道尺寸严重不匹配、重叠极差，几乎无法形成共振稳定结构！这使得硫酯反应物本身处于极高的能量状态，且硫醇基（-SR'）是极佳的离去基团（Good leaving group），一旦被水或无机磷酸 P_i 亲核进攻断开，生成的羧酸根（或酰基磷酸）获得巨大稳定能，从而释放出超过 31 kJ/mol 的自由能！

正因如此，在糖酵解的 GAPDH 反应中，酶先用活性中心半胱氨酸巯基（-SH）把氧化生成的能量捕获在高能硫酯中间物上，再由无机磷酸 P_i 进行磷酸解生成高能的 1,3-BPG；而在 TCA 循环中，琥珀酰-CoA 的高能硫酯键更是直接驱动了底物水平磷酸化生成 GTP/ATP！

四、焦磷酸（PP_i）水解推动合成、NTP 分工、腺苷酸激酶与 Atkinson 能荷调控

在本节最后，我们把 MIT 7.05 课堂推演与王镜岩第 16 章剩下的三大必考机制全部收网：

1. 焦磷酸（PP_i）水解驱动生物合成不可逆与“消耗 2 个高能键”算账铁律：
   在 L19 课堂末尾，Vander Heiden 教授总结道：当细胞进行需要极强热力学推力的聚合或活化反应——如 DNA 复制、RNA 转录、氨基酸活化合成氨酰-tRNA（蛋白质合成）、脂肪酸活化合成脂酰-CoA、乙酸活化合成乙酰-CoA，以及糖原合成中生成 UDP-葡萄糖（UDPG） 时，细胞绝不采用 ATP → ADP + P_i，而是采用腺苷酰基（或核苷酰基）转移策略，切下焦磷酸（PP_i）：
   第 1 步（活化/聚合）：底物 + ATP（或 NTP） ⇌ 活化底物-AMP（或延伸链） + PP_i (Δ G'^°₁ ≈ -14～ -45 kJ/mol)
   第 2 步（下游抽干产物）：PP_i + H_2O ──(无机焦磷酸化酶（Inorganic pyrophosphatase）)──► 2 P_i (Δ G'^°₂ = -33.5 kJ/mol 或 -19.2 kJ/mol)
   这里同时用上了热力学的两大法宝！通过遍布全身细胞的无机焦磷酸化酶将生成的副产物焦磷酸（PP_i）瞬间水解为两分子正磷酸（2 P_i），不仅额外叠加了约 -33.5 kJ/mol 的放能，更把第 1 步的产物 [PP_i] 浓度压低到接近于零，从而像拔河一样把大分子合成与底物活化反应彻底拉向不可逆的正方向！

• 华农能量计算题必考铁律：凡是发生 ATP → AMP + PP_i 的活化步骤（最典型的是脂肪酸 β-氧化前脂肪酸活化为脂酰-CoA，以及 2021 年真题考的乙酸活化为乙酰-CoA），虽然形式上只用了 1 分子 ATP，但因为断开了两个高能磷酸键（要把 AMP 重新充电回 ATP 必须经 AMP + ATP ⇌ 2 ADP 消耗两分子高能磷酸键），在计算净生成 ATP 数量时，必须按消耗 2 分子 ATP（扣除 2 个高能键）计算 2011/2012/2021/2024/2026·计算！

1. 四大核苷三磷酸（NTP）的精准专一分工（判断题高频陷阱！）：
   虽然所有 NTP 的标准水解自由能都与 ATP 完全相同（-30.5 kJ/mol），且可由核苷二磷酸激酶（Nucleoside diphosphate kinase, NDK）催化的可逆反应 NDP + ATP ⇌ NTP + ADP（Δ G'^° ≈ 0）随时由 ATP 充电转化，但它们在合成代谢中有着严格的专一领地：

• ATP：细胞通用的能量货币，直接驱动绝大多数激酶、合成酶、离子泵与肌肉收缩；
• GTP：专用于蛋白质生物合成（起始、延伸、终止因子）、糖异生（PEPCK 反应）以及 G 蛋白信号转导；
• UTP：专用于多糖/糖原的生物合成（活化葡萄糖生成 UDP-葡萄糖 UDPG） ⚑判断高频陷阱：误写为 GTP；
• CTP：专用于甘油磷脂与鞘磷脂的生物合成（生成 CDP-二酰甘油、CDP-胆碱、CDP-乙醇胺）。
  (记忆口诀：“A 通货、G 蛋白与异生、U 聚糖、C 磷脂”，另记植物合成淀粉用 ADPG！)

1. 细胞的终极保命机制：腺苷酸激酶、AMP 放大信号与能荷（Energy Charge）：
   在 L20 课堂上，Vander Heiden 教授提出了最后一个深刻问题：“如果细胞面临严重缺氧或饥饿，[ATP]/[ADP] 比值眼看就要崩盘了，细胞有没有最后一招紧急保命机制（Failsafe mechanism）来强行撑住 [ATP]/[ADP] 比值？”
   有！那就是由腺苷酸激酶（Adenylate kinase，在肌肉中又称肌激酶 Myokinase）催化的可逆歧化反应：
   2 ADP ⇌(腺苷酸激酶（Δ G'^° ≈ 0）) ATP + AMP

• 为什么这是绝妙的保命机制与最敏锐的能量探针？
  第一，通过把两个“半满电池” ADP 的高能磷酸拼在一起，既在分子上造出了 1 个急需的 ATP，又在分母上消灭了 2 个 ADP，从而在绝境中暂时保住了 [ATP]/[ADP] 比值！第二，在正常高能细胞中，[ATP] ≈ 5 mmol/L，[ADP] ≈ 0.5 mmol/L，而 [AMP] 极低（仅约 0.05 mmol/L）；一旦 ATP 仅消耗了 10%（降至 4.5 mmol/L，变化微不足道），生成的 ADP 经腺苷酸激酶转化后，会使细胞内的 [AMP] 浓度瞬間暴增 400%～ 600%！因此，在活细胞中，真正最敏感反映细胞缺能危机、被 AMPK（AMP 激活的蛋白激酶）、PFK-1 和肌糖原磷酸化酶 b 作为核心别构激活信号的，正是 [AMP]（或 [ATP]/[AMP] 比值）！
• Atkinson 能荷（Energy Charge, EC）定义与公式（华农 2020 年名解真题！）：
  为了用一个简洁的数值定量描述细胞内整个腺苷酸池（ATP + ADP + AMP）的充能状态，生化学家 Daniel Atkinson 提出了能荷的概念：
  能荷（Energy Charge, EC）：指细胞内总腺苷酸系统（ATP + ADP + AMP）中所含高能磷酸基团（以每个 ATP 含 2 个、每个 ADP 含 1 个、AMP 含 0 个有效可转移高能磷酸键计）的摩尔分数，是反映细胞能量状态并双向调节产能（分解）与耗能（合成）代谢途径的核心信号指标 2020·名词。
  其计算公式必须烂熟于心：
  能荷（EC） = ([ATP] + (1)/(2)[ADP])/([ATP] + [ADP] + [AMP])
• 取值范围与调控规律（见图 bioenergetics_coupling 右侧曲线）：当全部腺苷酸都是 AMP 时，EC = 0（电池彻底没电）；当全部都是 ATP 时，EC = 1.0（电池完全充满）。当细胞处于高能荷状态时，抑制产生 ATP 的分解代谢途径（R-产，如抑制 PFK-1、丙酮酸激酶、异柠檬酸脱氢酶），同时激活消耗 ATP 的合成代谢途径（U-用，如激活乙酰-CoA 羧化酶、糖原合成）；反之，当能荷下降时，强烈激活产能途径并抑制耗能途径！两条一升一降的陡峭曲线交汇之处，使绝大多数正常活细胞的能荷极其稳定地缓冲在 0.80～ 0.95（通常约 0.85～ 0.93） 的狭窄高能区间内！

五、迈向糖酵解的桥梁：碳原子的氧化还原阶梯与电子载体 NAD⁺/NADH

在 L20 课堂的后半段，Vander Heiden 教授完成了从生物能学通论向糖酵解（Glycolysis）的丝滑过渡。他问道：“既然我们要靠燃烧营养物来给 ADP 加上无机磷酸 P_i 合成 ATP，那么营养物里的能量究竟藏在哪里？”

答案是：藏在碳原子的还原态电子里！ 请看碳原子的五级氧化还原阶梯（从最还原到最氧化）：

-CH_{2-CH2-[烷烃（脂肪烃链，最还原）]} ──(-2e^-)──► -CH(OH)-_{[醇（糖类主体）]} ──(-2e^-)──► -CH=O / C=O_{[醛 / 酮]} ──(-2e^-)──► -COOH_[羧酸] ──(-2e^-)──► O=C=O_{[CO2]（最氧化）}

1. 为什么每克脂肪的热量（38 kJ/g，9 kcal/g）比每克糖或蛋白质（17 kJ/g，4 kcal/g）高出一倍多？
   因为氧气（O₂）是极强的电负性电子受体，碳原子每向氧转移一对电子（即氧化一级），就会释放大量自由能！糖类中的大多数碳原子已经连着一个羟基（处于半氧化的醇态，-CHOH-），而脂肪酸的长烃链则是完全饱和的烷烃态（-CH_2-，最还原态）且疏水无水合重量，因此在彻底氧化为 CO₂ 时，脂肪每个碳能脱下更多的电子、释放出高得多的能量！
2. 脱下来的两个电子（氢负离子 H^-）往哪儿放？——维生素 B₃ 与 NAD⁺/NADH：
   在糖酵解唯一的一步氧化反应（3-磷酸甘油醛脱氢酶 GAPDH 催化醛基氧化为羧基衍生物 1,3-BPG）中，底物醛基脱下了一个带有 2 个电子和 1 个质子 的氢负离子（Hydride ion，H^-，即 :!H^-）。细胞绝不能让游离电子乱跑，必须派出一辆专门装载电子的“分子卡车”——烟酰胺腺嘌呤二核苷酸（Nicotinamide adenine dinucleotide，氧化型记为 NAD⁺，还原型记为 NADH）！

• NAD⁺（及比它多一个核糖 2'-磷酸基的 NADP⁺）的活性功能部位是烟酰胺环（Nicotinamide ring），衍生自水溶性维生素 烟酸 / 烟酰胺（Niacin，即维生素 B₃ 或维生素 PP）。
• 反应机制：底物脱下的 2 个电子和 1 个 H⁺（即 1 个氢负离子 H^-）直接加成到 NAD⁺ 烟酰胺吡啶环的 C4 位上，使环上带正电的氮原子（N⁺）被还原为中性的 NADH，而底物脱下的另一个质子（H⁺）则游离进入水溶液：
  底物-H₂ + NAD⁺ ⇌(脱氢酶（Dehydrogenase）) 底物（氧化态） + NADH + H⁺
• 高频选择与判断陷阱：① NAD⁺、NADP⁺、FAD、CoA 分子内部都含有腺苷酸（AMP）结构，唯独 FMN（黄素单核苷酸）不含腺苷酸！② NADH 主要进入线粒体呼吸链氧化产生 ATP；而 NADPH 主要由戊糖磷酸途径产生，专供还原性生物合成（如脂肪酸、胆固醇合成），绝不是呼吸链的组成部分 2025·单选！③ 细胞内的 NAD⁺ 总量很少（催化量），如果在糖酵解中把 NAD⁺ 全都变成了 NADH 而不通过有氧呼吸或无氧发酵将其重新氧化回 NAD⁺，糖酵解就会在瞬间因缺乏 NAD⁺ 而彻底停摆！

1. 现代主流与华农近 10 年标准答案口径（Lehninger 8e / 王镜岩第 4 版 / 杨荣武）：1 分子线粒体 NADH → 2.5 ATP，1 分子 FADH₂ → 1.5 ATP（胞质 NADH 经 α-磷酸甘油穿梭［肌肉、脑］产 1.5 ATP，经苹果酸-天冬氨酸穿梭［肝、心］产 2.5 ATP）。按此计算：1 分子葡萄糖彻底有氧氧化产生 30 或 32 ATP（糖原中 1 个葡萄糖残基则为 31 或 33 ATP）；1 分子软脂酸（16C）彻底氧化产生 106 ATP 2008/2026·单选 2011/2012/2021/2024/2026·计算！
2. 老版教材口径（王镜岩第 3 版及早期参考书）：曾采用 1 NADH → 3 ATP，1 FADH₂ → 2 ATP（葡萄糖产 36 或 38 ATP，软脂酸产 129 ATP）。考场大题满分秘诀：凡遇能量计算大题，第一行先写明“按现代教材标准 1 NADH = 2.5 ATP, 1 FADH₂ = 1.5 ATP 计”，算出 30/32 或 106 ATP 后，顺手在括号里附注一句“（若按旧口径 3/2 计，则为 36/38 或 129 ATP）”——无论阅卷老师手里的评分细则是哪一版，你都能稳拿满分！

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🎧 MIT 7.05 课堂原声英文实录对照（本节关联 1 讲 · 完整逐段实录）点击展开 / 收起 ▾

🎬 MIT 7.05 Lec 20 · Bioenergetics & Glycolysis I (Prof. Vander Heiden)⏱ ~50:00

~00:00MITOCW | 20. Bioenergetics/Intro Pathways/Glycolysis I PROFESSOR:

~00:02Today we're going to discuss bioenergetics, continue that discussion and use that as a transition into also beginning to see how that applies to understanding glycolysis, the pathway that allows glucose breakdown. So just as a reminder of a couple of the points that we discussed last time, so last time we discussed what makes reactions favorable. And so remember, you could have any reaction, A to B-- A to B could be a single step. A to B could be an entire pathway, all steps in the pathway, that whether or not that reaction or pathway happens is determined by thermodynamics. It's not determined by enzymes. Enzymes are important, of course, to control the rate of reactions so they may allow things to happen that otherwise wouldn't happen. But whether the reaction happens or not is determined by free energy. And so free energy, delta G, remember we discussed is the following relationship. So it's related to this constant delta G0 prime, which is related to the equilibrium constant for any pair of products and reactants as well as this formula rt times the log of the products over the reactants. And so remember this means that whether a reaction happens depends on the equilibrium constant, but it also depends on the actual conditions present. That is how much substrate and how much product is there. And so what this means is that biology can come up with ways by keeping the product concentrations low to do things that may move in the opposite direction of what you would predict from equilibrium. And also by coupling these less favorable reactions to more favorable reactions, you can also allow biology to do unfavorable stuff. And this is, of course, where the role of ATP comes in. And we discussed this at the end of last time as well. Of course, what's important then becomes the ratio of ATP to ADP. And to illustrate that, we use this example. We use the-- remember ATP hydrolysis has a delta G0 prime of minus 7.5 kcals per mole. That, of course, tells you this is about the equilibrium constant. That tells us the equilibrium lies to the right. We can couple it to this first reaction in glycolysis, which traps glucose in the cell by adding a phosphate group. Delta G0 prime for this is positive 3.3 kcals per mole. This positive number tells you that the equilibrium lies towards the left. But when we couple these two reactions together, we add these two numbers together, and now we get a delta G0 prime of minus 4.2 kcals per mole, which are the coupled reaction. The equilibrium lies now again to the right. And that's how we can use ATP hydrolysis in order to carry out this unfavorable process, trapping the glucose in the cell.

~02:38Now how much glucose will be trapped? Well, of course, that will come from this reaction up here. And that's because whether or not a reaction occurs or not of course depends not on the equilibrium constant but on the actual conditions and the delta G of that reaction.

~02:54And so we can just plug that in for this system. And so we see delta G0 prime minus 4.2 kcals per mole. That says equilibrium lies to the right. But how far really depends on the conditions. And that's defined by this ratio of products over reactants. And you can imagine that this term here, as long as it's less than 4.2, you will favor glucose trapping inside the cell, whereas, of course, if it's greater than 4.2, then you'd favor-- the opposite direction would be favorable. Now this is really critical because this really explains how it is that ATP actually works to help cells do various reactions. And so if an ATP-ADP ratio exists such that it can make delta G less than 0 for the reaction that it's coupled to, that is how it provides the energy that then allows unfavorable reactions to occur. And so please do not make the common mistake that many, many biologists make when they state, oh, ATP equals energy. There's a lot of ATP. The cell must have a lot of energy. Remember it's the ratio of ATP to ADP that provides the energy. And so that means ATP can be sky high, but if ADP is equally high, there's no energy there. Consequently, ATP can be very low. But if ADP is extremely low, now you still have a high ATP to ADP ratio. And that is what provides the energy. And that's because this occurs regardless of concentration. It's because it's this ratio of ATP to ADP that ultimately says how much energy is there for the ATP to drive a reaction. In other words, delta G, or whatever the reaction is coupled to, is going to be proportional to the ratio of ADP to ATP. Great. Now how much ATP do you need then? What ratio do you need to drive a reaction? Well, it's hard to say that in absolute terms because it's going to be defined by how that ratio is coupled to the ratio of whatever other reactants and products. It also tells you, remember, as we talked about before, we can switch the directions of these reactions and we just flip the signs. The directions here are arbitrary. And so that means it also tells us how much energy we need to put in if we're going to charge up an ATP-ADP ratio. In other words, if we want to synthesize ATP, that is take ADP plus phosphate and make an ATP, well, what's this? What we just-- the exact opposite sign of that delta G0 prime is going to now equal plus 7.5 kcals per mole. And so that means that the equilibrium of this reaction is going to lie to the left just as that one lied to the right. And it's going to lie to the left by that much. And if we want to know how much energy it then takes to make ATP, well, obviously, it's not going to happen because equilibrium favors this direction. It's not spontaneous in that direction. But at various conditions, we can know by plugging in our formula delta G equals delta G0 prime plus 7.5 kcals per mole plus RT times the log of ATP. So if I have my ADP-- I'm sorry-- my ATP concentration sufficiently low relative to my ADP concentration, I can still make this reaction happen. Now that's not going to give you an ATP-ADP ratio that is actually consistent with being able to do anything else into the cell. And so but what it says is that if I am able to couple energy from nutrient metabolism to this reaction and overcome this positive 7.5 kcals per mole, that basically will allow me to now synthesize ATP and maintain an ATP-ADP ratio in the cell that allows cells to carry out other functions by the same rules that we talked about that would be unfavorable. Now there's lots of implications here for metabolism, because it says how much input you need to synthesize ATP. How much energy you need to synthesize ATP is going to depend on how charged this ratio is. And how useful that ratio is to catalyze other reactions is also going to depend on how charged that ratio is.

~06:50And in essence, this is why we cannot store energy as ATP. This is why we didn't eat ATP for breakfast and instead ate cereal that has a bunch of glucose in it, because we can use that glucose to get ATP but the ATP itself is not something that we really can burn to get energy in a sustained way. In other words, the delta G of ATP synthesis, making ATP is going to be proportional to the log of the ratio of ATP over ADP for all the reasons I described. The delta G of using ATP, ATP hydrolysis, using ATP to drive reactions is also proportional to RT times the log, in this case, of the ADP to ATP ratio. And so because you need to consume this ratio to get energy, and you need to put in energy to create this ratio, you are constant-- and how much is an exponential property-- you are constantly having to generate ATP in a way that allows you to then have a ratio that can then be consumed to fuel other reactions. And that's what's shown here on the slide. So I just graphed here what the ATP-ADP ratio is relative to a theoretical delta G that's shown here as a linear scale, here on a log scale, that basically there's this logarithmic relationship and so you need an exponential increase in energy input to drive this ratio up higher in order to, quote unquote, "store" more energy that can be used for later. And so this system is not one that you can use as an energy storage system and ultimately explains why cells have to do constant metabolism, have to constantly do some sort of metabolism to keep this ATP-ADP ratio charged for it to be in a useful range to have an ATP-ADP ratio that can be coupled to drive otherwise unfavorable reactions in our cells. So to say this another way, our cells need to constantly catabolize nutrients, burn glucose, burn some other fuel. They have to do this because they have to keep that ratio charged in a range where that can then support other cell functions, allow them to fight entropy and maintain order in biology. We alluded last time if you stop making ATP, well, what's going to happen? Well, that ATP-ADP ratio the cell has is going to fuel whatever reactions it can do until suddenly that ratio falls below the point where it's useful to drive those unfavorable reactions. In fact, if you stop ATP production, you will consume that ATP-ADP ratio in seconds. And this is why if you poison ATP production, cells die very quickly. Remember last time we mentioned you have a heart attack, blood flow stops going to a tissue. No more nutrients and oxygen. Cells die quickly. Cyanide poisoning does the same thing. Cells die quickly if you stop their ability to maintain this ATP-ADP ratio in the right range. And so remember the concentration, the absolute concentration of ATP for these reasons is not a good indicator of cell energy. The correct indicator of cell energy is the ratio of ATP to ADP. This term is sometimes referred to as the energy charge. And so that can be the ATP to ADP ratio, or oftentimes more correctly is the ATP to AMP ratio. Why the ATP to AMP ratio? Well, that's because once you understand this, you can now understand that cells have a failsafe mechanism to protect themselves if this ratio of ATP to ADP becomes too low. What is that mechanism? Well, you can carry out this reaction. Take two ATP molecules and turn them into transfer a phosphate to make an ATP plus an AMP. Why is that a failsafe mechanism? Well, if they care about this, if the ATP-ADP ratio is what maintains your ability to drive these thermodynamically unfavorable reactions, if I sacrifice the denominator by removing an ATP and get an ATP out of it, I've now at least temporarily fixed that ratio. Now this is something that obviously cannot go on forever.

~10:36But in the short term, it does allow cells to protect that ratio. And in fact, what cells actually defend is this ATPAMP ratio. And it's very, very difficult to change the ATP-AMP ratio in a cell and actually have it live. So it's really this ATP-AMP ratio that is most formally related to what would be called energy charge. But this is really why this is important is for all the reasons that I just described. All right. So hopefully, it's clear to you now why cells have to keep ATP-ADP ratio in the right range, why this is useful for them to now carry out all of these unfavorable reactions-- fight entropy, stave off death. And they can even have a failsafe here and sacrifice ADP as a way to try to maintain that as a very last resort. It also says why cells have to always do catabolism, because you always need the energy input to keep that ratio in the right range. However, you might imagine that, in real life, the messiness of biology, that there's lots of situations where the demand for ATP will suddenly increase. Well, of course, maybe you can just metabolize more sugar, do more nutrient catabolism to try to keep that ratio high. But that takes time. And so you need ways for cells to quickly respond to then allow them to mobilize all of this extra thing to-- extra fuel to do that. For example, we're walking around our ancestors on the plains somewhere and a lion appears. We don't have time to mobilize a bunch of new fuel. We have to be able to run away really quickly or the lion's going to eat us. And so cells have systems to suddenly respond to I need a lot more ATP demand to run away by having ways that one can buffer this ATP-ADP ratio, protect it in the short term, and allow cells time to ramp up nutrient metabolism in order to keep this high. All right. Well, if we wanted to create such a buffer system, how would we actually do it? Well, obviously, we have to follow all the rules of thermodynamics that we discussed. And we have to do so in a way that actually illustrates how it is that very well how metabolism works. In other words, if suddenly the ATP-ADP ratio drops, if we have a sudden drop in ATP-ADP ratio because we have increased demand, needing to run away from a lion, how could we actually create a buffer system that protects this? Well, to do it, we would need to couple ATP-ADP to some other product reactant pair, let's call them x and y, such that the ratio of y to x is coupled to the ratio of ATP to ADP. And you want it coupled in such a way that if that ratio goes down, this ratio can now protect ATP, basically favor ATP to ADP synthesis. However, when nutrients are plentiful and this ratio is really high, it then in turn can charge up this ratio so that it can buffer it later when the ATP-ADP ratio starts to fall. Now the most famous example of this actually comes from muscle physiology. And it's best described in terms of muscles and how they quickly respond to increased demand, like that running away from a lion. However, it in fact applies to many, many cells, including most non-muscle cells in animals.

~13:47All right. And I should say that it turns out that if you turn off ATP synthesis in a muscle, people have estimated that your ATP-ADP ratio will fall into a range where it is no longer useful to do anything within a matter of seconds, some claim even less than a second. And so this system ends up being very important.

~14:08Now the system that muscle uses, and many other cells do as well, is something called the creatine/creatine phosphate system. Now if you guys are weightlifters or bodybuilders out there, you probably have heard of creatinine because it's a very popular supplement that is taken for weightlifting, something I obviously don't do a lot of. But that creatine/creatine phosphate is out there. I'll describe to you what it actually does for muscle cells. And you can decide for yourself if this is actually useful as a energy or weightlifting supplement. So what is creatine/creatine phosphate? What does it look like? Well, it's basically looks like this. By the way, I will use this abbreviation often throughout this course, just a shorthand to draw a lot of these structures. So this here would be a carboxylic acid group drawn like this or like that, drawn to the carboxylic acid group just so you understand my shorthand. OK. So this here is the molecule creatine. If I put a phosphate here, this now becomes creatine phosphate. Again, just to remind you, this P with a circle over it is a phosphate group. I'll draw this particular phosphate group because it's a nitrogen-phosphate bond, a phosphoramidate bond. This is actually a bond that is more labile than the PO bonds that we're used to seeing in the phosphodiesters like ATP, et cetera. All right. So that's creatine and creatine phosphate. And if we draw out the reaction, creatine phosphate goes to creatine plus inorganic phosphate, so hydrolysis of creatine phosphate. This has a delta G0 prime of minus 10 kcals per mole. What does that mean? It's negative. Tells us about the equilibrium constant. Equilibrium lies to the right. Remember ATP to ADP hydrolysis shown over there, that hydrolysis has a delta G0 prime of minus 7.5 kcals per mole. Equilibrium also lies to the right. This number is more negative. This equilibrium lies further to the right than ATP hydrolysis does. So now what happens if we couple this reaction to the ATP hydrolysis reaction? So now we can do ATP plus creatine goes to creatine phosphate plus ADP. So we've coupled those two reactions together. So delta G0 prime, ATP to ADP is minus 7.5. Creatine phosphate to creatine in that direction is negative 10. In this direction, it would be positive 10. And so as I've drawn the reaction, this delta G0 prime will be plus 2.5 kcals per mole. 10 minus 7.5 plus 2.5, which tells us then that this equilibrium now lies to the left. However, which direction this reaction actually goes is not determined only by the equilibrium. It's really determined by delta G. So we can calculate that. So delta G equals delta G0 prime plus RT log of, as we've drawn it, creatine phosphate over creatine and ADP over ATP. So you can now imagine that this term here is the sum of those two. So it's plus 2.5. And so if this term is less than 2.5, what does that mean? Well, if it's-- I'm sorry. Less than negative 2.5, then delta G will be negative and you will favor creatine phosphate production. If this term is greater than negative 2.5, then delta G will be positive and you will favor ATP production. Why is that important? Well, if you think about the conditions, how you could make this term less than 2.5 or less than negative 2.5 or greater than negative 2.5, basically, what's going to happen is that if the ATP-ADP ratio is high, lots of energy around, no problems charging up, lots of things to use, charge up this ATP/ADP to a very high ratio, well, now you will favor this direction, creatine phosphate synthesis.

~17:33However, if ATP/ADP begins to fall and is lower, well, now this term is going to flip and you'll now favor ATP production. And so basically, by charging up this creatine/creatine phosphate ratio, it now creates a situation where it can buffer that ATP/ADP ratio so that when times are good, you charge up the buffer. When times are bad, you then consume the buffer in a way that allows you to protect your ATP/ADP ratio for a short time. And this is illustrated very nicely here in this slide that basically shows what happens in muscle physiology. And so this is just a theoretical curve that if you suddenly need to contract your muscle and you stop ATP production, well, ATP levels will fall. That ATP/ADP ratio more correctly will fall rapidly within the cell. Creatine phosphate will then now kick in to protect that ATP/ADP ratio as you can ramp up either anaerobic or aerobic metabolism to now buffer that ATP/ADP ratio and keep that muscle functioning. Now I want to make one point because oftentimes biochemistry textbooks will refer to things like ATP or creatine phosphate as, quote unquote, "high energy compounds." And of course, I guess they are high energy because they have these labile phosphate-nitrogen or phosphate-oxygen bonds. And because they're labile, hydrolysis is favorable. And to a certain extent, it's that hydrolysis that is providing energy. But remember it's not the metabolite or the bond in isolation that's really providing the energy. It's these ratios of ATP to ADP, creatine phosphate to creatine, that allow you to couple, for all the reasons we've spent now a lecture and a half discussing, two other reactions that really provides biological energy. All right. Now we're ready to discuss how it is that we can use glucose oxidation, nutrient catabolism, to keep ATP/ADP high, that is in a useful range, where it can allow cells to do otherwise thermodynamically unfavorable things. So how could we make this happen? Well, just like this example with creatine and creatine phosphate, if we want to use nutrient catabolism to do the same thing, we need to couple reactions of glucose oxidation, nutrient catabolism, that will allow us to synthesize ATP, ADP to ATP, and have that synthesis be favorable despite the high ATP/ADP ratio in cells. So let's think about it. So what is this? Nutrient catabolism. What did we talk about last time? Oxidizing glucose to carbon and CO2. So here's our reaction for that. So here's carbohydrate, glucose CH2O6 plus six O2 goes to six CO2 plus six H2O. That's the chemical formula for burning wood. So delta G0 prime for this is minus 686 kcals per mole. Quite a negative number. Equilibrium greatly favors the completely burned state, oxidized state, of that carbohydrate. And so you can imagine that this equilibrium is so favorable to the right, if we couple this reaction to ATP synthesis, you're clearly going to have plenty of energy there in order to maintain that ATP/ADP ratio in the right range to be compatible with cells using it to do other things. All right. So how do we actually do that? Well, if we do it, we have to follow the same rules of thermodynamics and do the same things that we did before. Basically, we have to do this reaction in a way that we can draw out a similar type of equation as we did for creatine and creatine phosphate. And at the highest level, that is how nutrient catabolism will allow us to keep ATP/ADP ratio high even as ATP hydrolysis is constantly fueling all these other unfavorable processes in the cell.

~20:55Now if we did this all in one step, our burning wood example, lots of heat is generated. That's too much energy. Not so good for biology. But rather what biology does, as we alluded to last time, is that because what we talked about in the beginning, that all of the things that we discussed are true for individual reactions or many, many reactions, basically, if we turn A into B and we do it in one step, like burning wood, or we do it in 500 steps, the thermodynamic consequences are exactly the same. And so what we need to do is do stepwise catabolism of glucose and take advantage of the fact that that delta G change is the same whether it's one step or many steps and in that stepwise fashion build in reactions where we create intermediate chemistries that allow us to do things like we did with creatine and creatine phosphate such that we can favor ATP synthesis when ATP/ADP ratio is in the physiological range. And in essence, this is the magic of how biology does this and basically couples nutrient catabolism in order to provide energy. And so the pathway that we're going to discuss today and in the next lecture is something called glycolysis, which simply put is glucose lysis. It's the stepwise chemical oxidation of glucose, which we know from our burning wood example is favorable. And we're going to use this pathway that basically will be a pathway to allow the stepwise lysis of glucose, a favorable reaction, to individual steps that set it up in a way such that ADP to ATP synthesis is favored despite a physiological ATP/ADP ratio. Now I want to point out that this really illustrates, back to that high energy compound thing, that ATP is not the only energy currency in the cell. In fact, glucose is much more energy for a cell than ATP. You get much more energy released from burning wood than you get from hydrolyzing ATP. And in essence, this is why we use glucose-- why nature has used glucose, and other molecules like it, as an energy storage device, because here is a relatively stable molecule, can sit around for a long time, and you can burn it when you need it to keep your ATP-ADP ratio high. All right. So, at the level of reactions and pathways now, how can we use glucose oxidation and couple it to drive ATP synthesis? Well, hopefully it's very clear that we have to basically set up a situation like this creatinecreatine-phosphate system that we already discussed. And so I want to start by discussing, what are the reactions in glycolysis that ultimately behave like this and allow this favorable ADP-to-ATP synthesis? And then we will go ahead and consider how we can make those reactions work in the context of an entire pathway that allows glucose oxidation. All right. Now, like creatine, phosphate, and creatine, both of the steps in glycolysis that favor ATP synthesis involve intermediates where you have phosphate on some molecule, plus ADP goes to ATP plus the phosphate coming off that molecule. Now, if we go to our creatine-creatine-phosphate system, obviously this equilibrium needs to lie to the right. And so the first one of these is as follows. It involves this molecule, phosphoenolpyruvate, also referred to as PEP. You will see throughout the course that we love to give these little acronyms to our metabolites because it's hard to constantly say and write out phosphoenolpyruvate. Much easier to say PEP. So phosphoenolpyruvate plus ADP goes to ATP. Plus, if I take the phosphate off of there, I get this molecule. This molecule is enolpyruvate. Enolpyruvate can be rearranged as follows. To make this molecule a ketone pyruvate.

~24:24You'll note-- hopefully remember from your organic chemistry classes that ketones like pyruvate can exist in two forms. They can exist in the ketone form or the enol form, rearranging via the chemistry that I've shown. The keto form is greatly favored over the enol form. However, by having a phosphate trap pyruvate the enol form-phosphoenolpyruvate, when you remove the phosphate and it's wanting to exist in the keto form, greatly enhances the energetics of this-- basically makes the reaction, the equilibrium want to lie very far to the right. And by the way of chemical tricks like this and others like it, you will see used over and over in biology. In fact, this is illustrated, if we asked what's delta G0 prime for this reaction, it's minus 15 kcals per mole. That tells us that the equilibrium of this reaction lies to the right. In fact, it lies far to the right, and in fact, this is-- you may remember ATP hydrolysis is negative 7.5 kcals per mole. So lies twice as far to the right as ATP hydrolysis does. And in fact, this minus 15 kcals per mole is the most favorable, at least in terms of individual reactions that at least I know of in metabolism. All right. Well, if this equilibrium for this is PEP to pyruvate, this here is delta G0 prime for ATP-ADP. So if we want to know what's delta G0 prime for the entire reaction, well, if PEP to pyruvate is minus 15, ATP to ADP is minus 7.5. Well here, we're going in the opposite direction, so it'd be plus 7.5. So it's minus 15 plus 7.5, means that for the entire reaction, this is minus 7.5 kcals per mole.

~26:00Means that this is favorable, equilibrium lies to the right. If we drew out-- I'm not going to do it again because I've done it a lot, but if we redrew out this equation, replacing pyruvate and PEP in the correct places, there for creatine, phosphate, and creatine, you would see that there's lots of conditions within cells that will favor ATP synthesis even when ATP-ADP ratio is high. All right. This reaction is catalyzed by an enzyme called pyruvate kinase. And pyruvate kinase is a classic example of a, quote-unquote, "irreversible reaction." However, I want to point out that the enzyme pyruvate kinase is actually named for the reverse reaction that's supposedly irreversible. Pyruvate kinase. If you take pyruvate and you act on the kinase as you learn from Professor Yaffe, that phosphorylates something. You phosphorylate pyruvate, you get phosphoenolpyruvate pyruvate kinase. And so remember that absolutely no reaction is reversible. It's only reversed-- what they mean by irreversible when they say pyruvate kinase step in glycolysis is irreversible, is that it is effectively irreversible under the conditions that are compatible with life that exist in cells. Of course, if you have very low ADP and very high ATP, you can come up with a way to net synthesize PEP. You just can't drive the reverse reaction under the ATP-ADP and the PEP pyruvate ratios that physiologically exist in cells. However, this is set up in a way such that you can favor ADP-to-ATP synthesis by converting PEP to pyruvate in a pathway, as we will see happens in glycolysis. All right. Now, the second reaction that I want to-- that allows ATP synthesis in glycolysis is catalyzed by an enzyme called phosphoglycerate kinase. And this carries out the following reaction. OK. So this molecule is 1,3-Bisphosphoglycerate, abbreviated 1,3-BPG. And basically the phosphate is transferred from here to ADP, which gives you this molecule, 3-phosphoglycerate or 3-PG.

~27:44So this reaction, the delta G0 prime for the entire coupled reaction, both steps of the reaction, is minus 4.5 kcals per mole. Tells you about the equilibrium constant. Equilibrium lies to the right, doesn't lie as far to the right as the PEP plus ADP goes to ATP plus pyruvate reaction, but nonetheless, is one where there's lots of conditions that are favorable in cells to maintain a high ATP-ADP ratio, although we will see later that this reaction actually runs quite close to equilibrium in cells, so there's actually conditions where it can go in the opposite direction as well, and we'll get to that later in the course. I want to point out one thing about this, one thing that makes this reaction favorable, as you notice here that this is a phosphate on an acid group, a so-called acid anhydride. And this is a pretty good phosphate donor. And so you want to lose that phosphate, which is one of the reasons why this reaction is favorable for ATP synthesis even at high ATP-ADP ratios. All right. So these are the two reactions in glycolysis that can favor ATP synthesis at a high ATP-ADP ratio just like creatine and creatine phosphate. And so hopefully now it's become apparent to you that if during our pathway of glucose catabolism we can build a pathway that couples what we already know is favorable, glucose oxidation, to generate these intermediates, PEP and 1,3-Bisphosphoglycerate, lacerate we can then build a pathway where those reactions can make synthesis of ATP favorable even though there's a high ATP-ADP ratio in cells, and in fact, maintain that high ATP-ADP ratio in cells. And in essence, this is the logic or the goal, if you will, of glycolysis and why it's important to provide energy to cells. OK. All right. Now, if we're going to make a pathway where glucose oxidation is coupled to making PEP and 1,3Bisphosphoglycerate, it's obvious we need to add phosphate to the system. There's no phosphate molecule on glucose. Now already mentioned earlier-- and I just erased it-- that to trap glucose in the cell, the first step in glycolysis, you add phosphate. But that phosphate donor is ATP. And so ATP being the phosphate donor doesn't help us here. If we're going to use phosphate transfer here, we need to have phosphate added into the system if this is going to work. We can't have ATP as our phosphate donor. You also saw lots of reactions from Professor Yaffe that involved phosphate transfer reactions. But again, ATP-- or GTP is the phosphate donor. And so for this system to work, we need a way to get inorganic phosphate onto a carbohydrate or we can't make things like PEP or 1,3-Bisphosphoglycerate that will allow ATP synthesis. Now we already saw earlier that additional phosphate to glucose is not favored. Remember, delta G0 prime of phosphorylating glucose was plus 3.3 kcals per mole. And it turns out that says equilibrium lies to the wrong side, so we can't net do that without ATP or energy input. And it turns out this is true for other sugars as well. And so you need energy input to add phosphate to the sugars. Plenty of energy input from glucose oxidation, but we need a reaction where phosphate addition is coupled to another reaction that makes it favorable, and we have to do this without making ATP. And again, it illustrates, again, the biological energy is not just about ATP. At a deeper level It's something different. And so to really discuss this and understand it, I need to introduce two concepts that are going to come up again over and over again in this class and across metabolism. The first of these is a very high-level bioenergetic concept, although it's really an energetic concept, and it comes back to what we already talked about.

~31:19So, we know burning wood is favorable. The CN H2O N plus oxygen going to CO2 plus water. That's burning wood, lots of energy released. Drew it up there for a single-glucose molecule and what the energy release the delta G0 prime is for that reaction. Now I want to point out chemically what is going on in this reaction. Chemically what is happening for this to occur is that electrons from the carbon are being moved to the oxygen. You are changing the oxidation state of carbon and of oxygen in order to carry out this reaction. Oxygen is a better electron acceptor than carbon, and so transferring those electrons from the carbon to the oxygen is favorable. That is, in essence, what is going on in this very favorable reaction to burn wood. Let me illustrate that a bit more explicitly here. So the carbon-- most of the carbons in carbohydrates are alcohols. So here, you can call this our carbohydrate, with the exception of the ketone or the aldehyde if it's a ketose or an aldose. All the other carbons in the sugar are alcohols. All the other bonds there. And so if we oxidize that carbon-- that is, if we remove electrons from the carbon, how can we do that? So this here generates this H- hydride ion. This is, in essence, two electrons that we are removing from the carbon. That gives us a ketone. So if we oxidize the alcohol and we remove two electrons, we get the ketone. We could oxidize this ketone further. That will give us the carboxylic acid. If we oxidize it even further, now we get CO2. So, moving in this direction, this is oxidation. Transferring electrons from the carbon to the oxygen to go in this oxidation direction, that is glucose oxidation is what's happening when we're burning wood. It's what happens when we burn glucose. Energy is released. This is largely what catabolism is about in biological systems. Now then reasons, then, that if we're going to move in the opposite direction, so-called reduction, that is going in this direction. Reduction. That's largely energy storage or anabolism in metabolism. We are storing energy that we can burn later. And of course, what's the more extreme version of this? Well, what if we add two more electrons to the carbon going in this direction? Well, what do we end up with? Well, now we end up with the saturated carbon. more reduced than the alcohol is the fully saturated carbon. You will see later that this is what fat is. You saw that in some of the earlier lectures. It's also what gasoline is. What's gasoline? It's fully saturated hydrocarbons that we then burn to CO2 and derive energy. Just like burning wood, burning gasoline all in one step, oxidizing the gasoline gives you energy. There's more energy in gasoline. You can release more energy per weight from gasoline, from fully-saturated hydrocarbons than you can from wood. Same thing happening here. What has more calories in your food? Everyone knows fat has more calories per weight than sugar. It's because the more reduced our carbon is, the more energy it stores, and as we oxidize that carbon, that is what releases the energy. And so energy transduction in biological systems-- and actually, in non-biological systems, as I just illustrated with the wood and gasoline example, but definitely in biological systems, is largely about oxidation and reduction reactions, and you are going to see this coming up over and over again.

~34:34Now, based on what I just said, the real magic, if you will, of glycolysis can be further refined to say that what glycolysis does is it couples the favorable oxidation of carbon with reactions that allow phosphate additions and production of intermediates, like PEP and 1,3-Bisphosphoglycerate, where synthesis of ATP is favored despite the high ATP-ADP ratio in cells. If you think about it, that process then allows you to keep that ATP-ADP ratio in the range where it can then be useful for all the reasons we've been talking about now for two lectures. Allows the cell to couple that ATP-ADP ratio, that-- in ATP hydrolysis to otherwise carry out otherwise unfavorable reactions. OK. So, based on that, arguably the key, or at least a very important reaction in glycolysis, is the oxidation step which happens to add the phosphate and also happens to make 1,3-Bisphosphoglycerate, which we saw earlier can be used to drive ATP synthesis. And so that reaction is the following. So this molecule hopefully looks familiar to you from the carbohydrate lectures. This is D-glyceraldehyde 3phosphate. I will abbreviate it frequently is G-3P for glyceraldehyde 3-phosphate, but it's D-glyceraldehyde 3phosphate. Remember, it's a D, sugar, because the OH group is pointing to the right as we drew it this way. It's also an aldose because it's a aldehyde sugar, and it's phosphorylated on the 3 position-- remember the way we numbered sugars was starting at the carbonyl-- 1, 2, 3. This is carbon-3, phosphorylation on the 3 position. Glyceraldehyde 3-phosphate. OK. So, this step generates 1,3-Bisphosphoglycerate. Well, to do this, there's actually two things that happened here. First we had to add the phosphate to this. It says two phosphates on it. bisphosphoglycerate, this has a single phosphate. So inorganic phosphate has to be added to generate that molecule. By the way, adding that phosphate delta G0 prime is positive plus 1.5 kcals per mole. That means even equilibrium, this reaction would lie to the left, of course, because delta G0 prime is positive. However, remember the next step downstream of this is the one shown over here where we do ATP synthesis. That step is very favorable. So this is an example where metabolism has coupled a very favorable step to a step that's less favorable. That can keep the 1,3-BPG levels low and pull the entire system forward, the metabolic trick we discussed in the last lecture. OK. You'll note the other thing that's happening here, other than adding that phosphate group, is that we've changed this aldehyde to a carboxylic acid. That is an oxidation reaction. Ketone to the acid is an oxidation reaction. I will illustrate over here this so you can see why it's an oxidation reaction. So here's our aldehyde. If we take electrons from the carbon and that generates this hydride ion to electrons, what are we left with? Well, we're left with this intermediate that will never exist with a positive charge on the carbon. Water, which, of course, is abundant in biological systems. And then come-- OK. And it basically illustrates that as I go from the aldehyde to the acid, of course, I make a proton and a hydride ion, but really, I have changed the oxidation state of the carbon from the ketone to the acid state. Two electrons are lost there, is shown as that hydride ion. OK. So, what does that mean? Well, that means that we have to maintain that balance in this reaction as well. So water is going to have to come in and a proton will be produced. But the key thing is we also generate two electrons, this hydride ion that have to go somewhere. Now, finding a place for those two electrons to go is what brings up the second high-level concept that I now need to spend some time discussing.

~38:08And this second high-level concept that I need to introduce is the role of cofactors, and cofactors also is effectively the role of vitamins and what role that these things play in metabolism. Most vitamins are used to support metabolic reactions, and we're going to learn all about what many of the vitamins that you guys are familiar with reading the side of your cereal box actually do throughout the course.

~38:31Now cofactors, as I said, often involve vitamins, are molecules that provide useful chemical groups that facilitate the reaction chemistries that are needed in metabolism. So they provide some useful chemical groups to facilitate some of the chemistries that we need to carry out metabolic reactions. They are, in general, in small non-stoichiometric quantities in order to carry out this function. What do I mean by that? Meaning that you can imagine that the cell will convert lots of glyceraldehyde 3-phosphate to 1,3Bisphosphoglycerate, but the enzyme, you don't need one enzyme for every time you convert that reaction. The same enzyme can do it over and over and over again. Well, it turns out, cofactors are much more like enzymes in that respect. They need to be recycled in a way that they can catalyze the turnover of reaction many, many, many times, and it'll be much clearer what I mean by this when we get into some of the details. Now, the vitamin concept comes from the fact that often these cofactors or parts of the cofactors are not made by animals, not made by humans. If they're not made by us, but we need them to do our metabolism, well now we have to get them from the diet, and this is really the concept of vitamins. These are small things that we have to get in quantities from the diet, and it turns out they're very important to help us carry out the metabolism we do. As an aside, the vitamin industry is a very big business. Lots of unfounded claims out there. I'm not going to get into-- that can get very political. I'm just going to say that just know that more is not always better, and the goal for this class is really to understand what some of these vitamins actually do. OK. Now, the relevant cofactor for the reaction that we're talking about is a cofactor called nicotinamide adenine dinucleotide, which is always abbreviated NAD or NAD+. This stands for nicotinamide adenine dinucleotide. You see why we want to abbreviate that NAD. And basically what nicotinamide adenine dinucleotide is is it's a factor that can solve our problem of where to put these electrons in this oxidation reaction. That is, you can accept two electrons from a reaction like this one here and cycle to another form of the cofactor called NADH. All right. What is nicotinamide adenine dinucleotide? Well first, let's draw what it looks like. OK. Sorry it's squeeze down here a little bit. So this here is adenine. This group up here is nicotinamide. So if you look at this, this is a dinucleotide. And so down here, this, of course, is, from here down, is AMP. That phosphate is now over here. This is a separate nucleotide, where rather than having one of the AGCTU bases that you're used to from RNA and DNA, nicotinamide is the base. And so it's this AMP plus this nicotinamide base is a monophosphate, or a nicotinamide adenine dinucleotide. This, the nicotinamide group, is derived from a vitamin called niacin. And so from the side of your cereal box, this is what niacin does. It allows you to generate the nicotinamide group that's part of this cofactor, nicotinamide adenine dinucleotide. Now, nicotinamide adenine dinucleotide is useful because of what that nicotinamide group can do.

~41:36And so I'm not going to draw out the whole molecule, but I'll show you. So NAD+, which is the oxidized form of nicotinamide adenine dinucleotide, is as I drew it up there. So this here would be attached to the rest of the dinucleotide. So this is the oxidized form. I can have-- here's my hydride ion, which, remember, is two electrons. If I carry out that chemistry to add those two electrons to the nicotinamide group, you now end up with the reduced form of nicotinamide adenine dinucleotide NADH. So the difference between NAD+ and NADH, it's the H, but it's really the two electrons that were added in adding that H. So it's oxidized form and reduced form. So remember, I want to stress this again, at least in biology we can't create or destroy matter. You can do that in nuclear physics, but we can't do that in biology. And so that means that if we do an electron transfer like we did in this reaction where we oxidized the aldehyde in glyceraldehyde 3-phosphate to the acid in 1,3Bisphosphoglycerate, those electrons have to be accounted for. And so if one molecule is oxidized, another one has to be reduced. So redox reactions always-- that is, redox, oxidation-reduction reactions, always have to happen in pairs. And so over there we have the aldehyde in glyceraldehyde 3-phosphate is oxidized to form the acid in 1,3-Bisphosphoglycerate. As part of that reaction, we can couple it such that the nicotinamide group, which is oxidized in NAD, can be reduced to the nicotinamide group that is found in NADH. So that means we can draw this in here, is that having that cofactor coupled to this reaction now allows us to fix the redox problem of this reaction. Now this reaction here is catalyzed by an enzyme called glycerol 3-phosphate dehydrogenase. I'll draw that out in the second. Often abbreviated GAPDH, the loading control in all of your western blots, is the enzyme GAPDH, glyceraldehyde phosphate dehydrogenase. That is the enzyme that carries out that reaction. Now notice, the name fits. It's a dehydrogenase, it's removing a hydrogen, removing two electrons as a hydride ion, but it's really-- what's important here is the electrons moving, not the hydrogen itself. So in that way it's a little bit misleading. And you will see that in general, dehydrogenases are classes of enzymes that catalyze oxidation-reduction reactions, and as a result, often involve NAD/NADH as cofactors because they facilitate those electron transfer reactions. OK. So now we're able to describe how GAPDH works in great detail. And so the chemistry of GAPDH works as follows. And so in the active site of GAPDH, there's a cystine residue. That cystine residue has a sulfur shown here. Also bound near the active site is in NAD molecule to catalyze the reaction shown. That NAD has to be in the oxidized state, so it's NAD+. And so glyceraldehyde 3-phosphate binds in the active site. There's glyceraldehyde 3-phosphate. By the way, I will show you how many reactions in metabolism work chemically. Obviously we don't have time in this class, an overview class to go into all the great chemical details of what's going on and exactly how everything works, but I'll at least try to give you a flavor of some of the chemistry that happens throughout the course. All right. So once you have, then-- OK. So now you have this intermediate bound into the enzyme active site. You can now carry out the oxidation of that carbon. So these are two electrons being removed as a hydride ion. They can be transferred to NAD in the active site. That generates NADH by the exact chemistry that I just showed you. OK.

~45:03That NADH now needs to be exchanged out or the enzyme will not be able to catalyze the next series of reactions, and that NADH will then go somewhere else, and of course, has to be cycled back to NAD, but that doesn't happen in this reaction, per se. OK. Next, this here, you'll notice, is that by the way this chemistry worked, we formed this thioester bond in the active site. This is another thing that you will see over and over again in metabolism. This sulfur in the thioester bond is a good leaving group, and so it's really setting up a situation that will now help with the phosphate addition. So here, I'll draw out inorganic phosphate. And that, of course, will give us the identical enzyme active site that we started with plus 1,3-Bisphosphoglycerate. And that 1,3-Bisphosphoglycerate can now be coupled to ATP synthesis to make 3-phosphoglycerate. And so you can see, in essence, what this reaction has done is that it has coupled oxidation of the aldehyde to the acid, the aldehyde in 3-phosphate to the acid in 1,3-Bisphosphoglycerate, which can then do phosphorylation of ADP to make ATP. So in essence, this is oxidative phosphorylation. Now I realize in high school or in other classes, oxidative phosphorylation means something different to you, and of course, we will discuss the process that is called oxidative phosphorylation in great detail later in this course. But I want to point out that the chemistry that's actually happening here in glycolysis is also oxidative phosphorylation. It's basically how you coupled this favorable carbon oxidation that's occurring at this GAPDH step in a way that allows you to synthesize ATP at a high ATP-ADP ratio, which in the end will allow cells to do otherwise unfavorable things. OK. Now we're ready to discuss these reactions, the GAPDH reaction, the pyruvate kinase reaction, the phosphoglycerate kinase reaction, and how they fit into glycolysis as a metabolic pathway in a way that allows cells to use glucose metabolism-- that is, the breakdown of glucose, the burning of glucose as a way to get energy. And that pathway is, of course, glycolysis or glucose lysis that we mentioned earlier. Glycolysis is a very ancient and ubiquitous pathway. It's used by essentially all life on Earth and probably has been for at least 2 to 3 billion years. Glycolysis, I guess, as a pathway was, quote-unquote, "discovered" mid-19th century by Louis Pasteur, and Louis Pasteur, of course, described life, microorganisms as being responsible for the process of fermentation. That is, conversion of glucose into alcohol, something many college students are interested in, as well as other organic acids. And of course, fermentation to make alcohol or to store other types of organic acids has been used in food preservation for centuries, which is really why Pasteur's discoveries were so impactful at the time. Now even though Pasteur discovered this, he didn't really understand chemically how it worked, and it actually took decades to figure that out. And when it was finally figured out and described as the pathway called glycolysis, this was done by two chemists, two chemists in Germany, Embden and Meyerhof. And basically what they did is they pieced together several enzymatic or chemical activities that have been purified from cell lysates mostly from yeast and used those to build a pathway that described chemically how you could start with glucose and chemically break down that glucose in a way that made sense in this fermentation pathway, and that is why glycolysis is sometimes called the Embden-Meyerhof pathway.

~48:20Now, glycolysis does not completely convert glucose to CO2. Instead, what glycolysis does is it converts glucose-so I will draw here in the pyranose form. It converts glucose by a number of steps into two molecules of the organic acid pyruvate, which, of course, was the product of that pyruvate kinase reaction that we showed earlier. Note that the fermentation that Louis Pasteur described was further metabolism of that pyruvate into something else. In the case of yeast, it was ethanol or ethyl alcohol, or something else. The fermentative product in mammals is this organic acid lactate. And what we will see next time is that this fermentation of pyruvate into one of these other molecules ends up being an alternative to the further oxidation of pyruvate to CO2 which requires oxygen, and the reason oxidizing pyruvate to CO2 requires oxygen is because ultimately we have to regenerate that NADH we made in glycolysis to allow that cofactor to cycle and glycolysis to keep moving. Fermentation gives us an alternative way to do this, which we'll discuss in great detail in the next lecture. But first I want to focus on glycolysis and what we can describe next time as really how we can build a pathway to turn glucose into pyruvate. This glycolysis, this Embden-Meyerhof pathway, and some of the details of that pathway are shown here on this slide. And this is where we'll start off the next lecture, discussing how each of these steps in glycolysis function in a way that allow a pathway to generate phosphoenolpyruvate, 1,3-Bisphosphoglycerate, incorporate this GAPDH reaction, and do so in a way that's overall energetically favorable to allow the cell to couple this oxidation of glucose to keeping ATP-ADP ratio in the right range to support other unfavorable functions in the cell. OK, thanks.

★本章小结：核心代谢逻辑链

1. 单糖的立体选择与环化保护逻辑：单糖是多羟醛或多羟酮（二羟丙酮唯一无手性碳；D/L 构型仅看离羰基最远的手性碳 C^*，与旋光方向无关）。在水溶液中，五碳以上单糖通过分子内羟基与羰基亲核加成形成稳定的六元吡喃环或五元呋喃环，产生新手性中心异头碳与 α/β 异头物（口诀“Haworth 上 D 顺 β”），并经微量开链形式互变产生变旋现象。自然界之所以选定 D-葡萄糖作通用血糖，是因为其主要存在形式 β-D-吡喃葡萄糖（占 ～ 2/3）在椅式构象中全部 5 个非氢大基团均处于平伏键（空间位阻最小），从而将 >99.9% 的活泼醛基死死锁在环内（开链 <0.024%），最大限度避免了非酶糖基化对细胞蛋白质的毒性损伤。
2. 糖苷键几何学与二糖/多糖的功能分野：半缩醛羟基成苷变为缩醛后即丧失开环能力。蔗糖 Glc(α1↔ 2β)Fru 与海藻糖 Glc(α1↔ 1α)Glc 因两个异头碳双双被糖苷键锁死而成为稳定的非还原糖（分别作植物筛管与昆虫血淋巴运输糖）；麦芽糖、乳糖 Gal(β1→ 4)Glc、纤维二糖则保留右端游离异头碳而具还原性。在多糖中，α(1→ 4) 糖苷键形成天然折角，盘绕成适合紧密储能的左手螺旋（直链淀粉、支链淀粉、糖原）；而 β(1→ 4) 糖苷键使相邻残基翻转 180^° 呈扁平伸展直链并交织成致密氢键网，铸就了植物细胞壁（纤维素）、昆虫外骨骼（几丁质）与细菌细胞壁（肽聚糖）的钢筋铁骨。
3. 糖原极限分支与加磷酸解的能量账单：动物以高度分支（每 8～12 个残基一个 α-1,6 分支）的糖原颗粒储能，既解决了游离葡萄糖会导致细胞渗透胀裂的危机，又创造出海量非还原端供糖原磷酸化酶同时并发切糖。更精妙的是，胞内糖原动员采用无机磷酸 P_i 进攻的加磷酸解（生成 G-1-P → G-6-P）而非消化道的水解，既用负电荷将糖磷酸酯锁在胞内，又省去了己糖激酶消耗的 1 分子 ATP（使每个糖原葡萄糖残基无氧酵解净赚 3 ATP，有氧彻底氧化产 31 或 33 ATP）。
4. 杂多糖防御网与细胞表面“糖密码”：细菌肽聚糖以 GlcNAc(β1→ 4)MurNAc 为主链（溶菌酶靶点）、以含 D-氨基酸的四肽及五聚甘氨酸桥交联（青霉素靶向转肽酶阻断新壁交联）。动物胞外基质由高负电荷的糖胺聚糖（GAG）与核心蛋白组装成蛋白聚糖（糖占 ≥ 95%；透明质酸唯一不硫酸化且不共价连蛋白，硫酸角质素唯一不含糖醛酸，肝素负电荷密度最高抗凝）。细胞膜糖蛋白经 N-连接（GlcNAc-Asn） 或 O-连接（Ser/Thr） 暴露寡糖链（如 ABO 血型抗原），由凝集素专一识别。
5. 生物能学热力学与 ATP/能荷调控闭环：酶只能加速平衡的到达而不能改变 Δ G'^°。生命对抗宇宙熵增、驱动 Δ G'^° > 0 反应的两大法宝是下游抽干产物（压低 [P]/[S] 使实际 Δ G < 0）与热力学偶联（加上强放能反应）。真正提供生物能量的是远离平衡的 [ATP]/[ADP] 比值而非 ATP 绝对浓度；ATP 因处于磷酰基转移势能阶梯正中间（-30.5 kJ/mol），上接 PEP（-61.9）、1,3-BPG（-49.3）、磷酸肌酸（-43.0，肌肉 ATP 缓冲池） 和高能硫酯键（乙酰-CoA -31.4） 充电，下通过基团转移（或切下 PP_i 水解耗 2 个高能键）驱动合成，并由腺苷酸激酶与 Atkinson 能荷（0.80～ 0.95） 实现全细胞产能与耗能的精密自动巡航。

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★名词解释速背卡（中英对照高频名解）

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★易混概念与新旧教材口径辨析

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★华农历年真题章末综合自测

一、名词解释（每题 3–5 分）

1. 结合糖（糖缀合物） 2025·名解 & 糖蛋白 2023·名解 & Proteoglycan（蛋白聚糖） 2026·名解
2. 能荷（Energy charge） 2020·名解
3. 激酶（Kinase） 2021/2022/2023·名解
4. 代谢组学（Metabolomics） 2019/2022·名解
5. 写出缩写中文全称：PEP 2013·名解

二、单项选择题与判断题（经典真题精选）

1. 【2026·单选】 下列糖类中具有 β-1,4-糖苷键的是（　）。
   A. 蔗糖　　B. 麦芽糖　　C. 乳糖　　D. 糖原
2. 【2024·单选】 蛋白质中哪种氨基酸可作为糖苷化位点？（　）
   A. Pro　　B. Lys　　C. Ala　　D. Thr
3. 【2012·单选】 有关葡萄糖磷酸化的叙述中错误的是（　）。
   A. 由己糖激酶或葡糖激酶催化　　B. 消耗 1 分子 ATP　　C. 磷酸化后的葡萄糖能自由通过细胞膜　　D. 反应在生理条件下不可逆
4. 【2008·单选】 糖代谢中间产物中含有高能磷酸键的是（　）。
   A. 6-磷酸葡萄糖　　B. 6-磷酸果糖　　C. 1,6-二磷酸果糖　　D. 1,3-二磷酸甘油酸
5. 【2025·单选】 （　）不是呼吸链的组成部分。
   A. NADH　　B. NADPH　　C. FADH₂　　D. Cytochrome c
6. 【2024·判断】 溶液中单糖链状和环状结构可相互转变，只有链状有氧化还原反应。（　）
7. 【2024·判断】 青霉素作用是抑制新细胞壁形成而不能破坏已经形成的细胞壁。（　）
8. 【2012/2018·判断】 Δ G > 0 表示某反应不能自动进行；如在酶的催化下，与之相偶联的放能反应则可驱动这类反应进行。（　）

三、填空题与简答/论述综合题

1. 【2012/2024/2025·填空综合】

• 直链淀粉遇碘呈（①）色，支链淀粉遇碘呈（②）色；糖原和支链淀粉结构十分相似，都由许多（③）组成，通过（④）和（⑤）糖苷键相连。
• （⑥）是人和动物餐间及肌肉剧烈运动时最易动用的葡萄糖贮库，一个原因是它含大量的（⑦）可被迅速动员分解。
• 纤维素是由（⑧）组成，它们之间通过（⑨）糖苷键相连；糖蛋白中能以 N-糖肽键与寡糖相连的氨基酸残基是（⑩），以 O-糖肽键相连的是（⑪）、（⑫）。

1. 【2019/2021·简答】糖在生物体中有哪些重要生物学功能？请分别举例说明。
2. 【2021·简答计算】计算 1 分子游离葡萄糖和 1 分子乙酸在细胞内完全有氧氧化成 CO₂ 和 H_2O 后分别净产生多少分子 ATP？（请写出折算依据与分步账单；若改为糖原中的 1 个葡萄糖残基，结果又有何变化？）
3. 【2022·简答】如何研究生物体内的某一条新陈代谢途径？

如果把细胞比作一座昼夜运转的繁华大都市，那么能量货币 ATP 就是市民手中随时花出去的现金。在 MIT 7.05 第 20 讲的开场白中，国际顶尖癌症代谢学家 Prof. Matthew Vander Heiden 向全班抛出了一个颠覆死记硬背的灵魂追问：“为什么我们早餐吃的是富含葡萄糖的麦片，而不是直接舀一碗纯 ATP 吃下去？”答案藏在热力学公式 Δ G = Δ G'^° + RT ln ([ADP][P_i])/([ATP]) 里——真正驱动生命逆熵而行的从来不是 ATP 的绝对分子数，而是细胞维持在远偏离平衡态的高 [ATP]/[ADP] 比值！一旦停止合成 ATP，人体肌肉或心肌细胞内的 [ATP]/[ADP] 比值在短短几秒钟内就会崩盘，细胞随即死亡。为了时刻给这个比值“充电”，地球上几乎所有生命在距今约 30 亿年前的无氧太古宙，共同演化出了一条最古老、最普适的碳水化合物拆解流水线——糖酵解（Glycolysis，又称 Embden-Meyerhof-Parnas 途径，简称 EMP 途径）。

站在有机化学与生物能学的第一性原理视角看，糖酵解绝不是十个孤立酶促反应的枯燥罗列，而是一场精妙绝伦的“华尔街能量套利交易”：细胞先在前 5 步（准备期）主动借贷并倒贴 2 ATP，把一分子稳定的六碳葡萄糖对称改造成两分子高活性的三碳醛糖（3-磷酸甘油醛，G3P）；紧接着在第 6 步（全途径唯一一步氧化反应），利用酶活性中心的半胱氨酸巯基与无机磷酸 P_i，将醛基氧化成羧基释放的巨大自由能“截胡”并锁进高能酰基磷酸键（1,3-BPG）中；随后在后 5 步（获能期）通过两次底物水平磷酸化连本带利收回 4 ATP，净赚 2 ATP 和 2 NADH。

更令人拍案叫绝的是，当细胞面对缺氧危机、剧烈冲刺或是癌变增殖时，这条古老通道展现出惊人的调控弹性：为什么无氧条件下必须把丙酮酸还原成乳酸或乙醇？核心根本不是为了造乳酸，而是为了把第 6 步积压的“电子废料” NADH 重新氧化回 NAD⁺，防止糖酵解因辅酶枯竭而瞬间停摆！而在第 21 讲中，亲自揭开肿瘤代谢之谜的 Vander Heiden 教授更会带你直击三大限速酶（HK/GK、PFK-1、PK）的别构与激素开关，并彻底讲透为什么癌细胞在氧气充足时依然疯狂抢糖产乳酸（Warburg 效应），甚至故意换用低活性的 PKM2 同工酶来为肿瘤生物量合成制造“战略性交通拥堵”。现在，就让我们推开 MIT 7.05 教室的大门，从化学键与自由能的底层逻辑出发，将糖酵解的每一个考点与机理彻底融会贯通！

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2.1能量电荷、磷酸肌酸缓冲与糖酵解准备期（第 1–5 步）：为什么赚钱前要先倒贴两分子 ATP？

一、从 [ATP]/[ADP] 比值到磷酸肌酸缓冲池：遭遇狮子时肌肉如何撑过前 3 秒？

在正式拆解葡萄糖之前，Vander Heiden 教授在黑板上写下了 ATP 水解反应的热力学方程，纠正了生物学初学者最容易犯的一个概念错误：细胞内提供自由能驱动吸能反应的，绝不是 ATP 的绝对浓度，而是远高于平衡态的 [ATP]/[ADP] 浓度比值。哪怕细胞里的 ATP 浓度高上天，只要 ADP 浓度同样高（处于化学平衡态，Δ G = 0），这套系统就做不了任何有用功；反之，因为生成 ATP 所需的自由能与 ln([ATP]/[ADP]) 成正比，想要把能量以超高 [ATP]/[ADP] 比值的形式“储存”起来，需要的能量投入会呈指数级暴增。这就是为什么生命不能靠囤积 ATP 来过冬，而必须把能量储存在化学性质稳定、还原度高的葡萄糖和脂肪里，再通过每分每秒不间断的分解代谢（Catabolism），像踩水一样动态维持高 [ATP]/[ADP] 比值。

那么，如果我们的祖先在非洲草原上散步时突然撞见一头狮子，全身骨骼肌瞬间需要海量 ATP 来狂奔逃命，而动员糖原分解和加速糖酵解需要几秒钟的响应时间，肌肉细胞如何在不到 1 秒的时间内防止 [ATP]/[ADP] 比值崩塌？大自然设计了两道极其聪明的防线：

1. 腺苷酸激酶（Adenylate Kinase）的保底机制与能荷（Energy Charge）：当 ATP 被大量消耗导致分母 ADP 激增时，细胞内的 腺苷酸激酶（又名肌激酶）可催化可逆反应：2ADP ⇌ ATP + AMP（Δ G'^° ≈ 0）。这一招通过牺牲两个分母（ADP）换回一个分子（ATP）和一个 AMP，不仅在危急关头短期保住了 [ATP]/[ADP] 比值，更让细胞内本底浓度极低的 AMP 浓度发生数十倍的剧烈飙升！因此，在细胞生理学中，[ATP]/[AMP] 比值（或阿特金森能荷 Energy Charge = ([ATP] + 0.5[ADP])/([ATP] + [ADP] + [AMP])）才是反映细胞真实能量状态最灵敏的指针，而 AMP 也順理成章地进化成了激活糖酵解限速酶（PFK-1）最核心的“低能警报器”！
2. 肌酸 / 磷酸肌酸（Creatine / Creatine Phosphate）高能缓冲系统：在脊椎动物骨骼肌、心肌和脑组织中，还配备了一个专门缓冲 [ATP]/[ADP] 比值的“高能蓄水池”——磷酸肌酸（creatine phosphate / phosphocreatine，简称 CrP）：肌肉和脑组织中储存高能磷酸键的化合物，含有一个比 ATP 磷氧键更不稳定的磷氮键（phosphoramidate bond，Δ G'^° = -43.1 kJ/mol 即 -10 kcal/mol），可在肌酸激酶（CK）催化下迅速将磷酸基团转移给 ADP 生成 ATP。
   磷酸肌酸（CrP） + ADP + H⁺ ⇌(肌酸激酶（CK）) 肌酸（Cr） + ATP (Δ G'^° = -12.6 kJ/mol 或 -2.5 kcal/mol)
   当你在安静休息、营养充足时，细胞内 [ATP]/[ADP] 比值极高，根据质量作用定律，Δ G = Δ G'^° + RT ln ([CrP][ADP])/([Cr][ATP]) < 0，反应逆向进行，把多余的磷酸基团充入磷酸肌酸池；一旦狮子扑过来，肌肉剧烈收缩导致 [ATP]/[ADP] 刚一抬头下降，反应立即正向倾泻，在毫秒级时间内把 ADP 重新磷酸化为 ATP，为糖酵解的全面启动争取宝贵的黄金几秒钟！

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二、糖酵解（EMP 途径）全景鸟瞰与前 5 步“准备期”的化学精算

当磷酸肌酸帮细胞顶住最初的几秒后，真正的能量主力军——糖酵解（Glycolysis）必须立刻接管战场。19 世纪中叶，路易·巴斯德（Louis Pasteur）发现微生物能在无氧条件下将葡萄糖转化为乙醇或有机酸（发酵）；此后历经数十年，德国生化学家 Густав Embden、Otto Meyerhof 与波兰生化学家 Jakub Parnas 从酵母和肌肉提取液中一步步拼出了完整的十步酶促反应拼图，因此糖酵解（glycolysis / EMP pathway）：又名 Embden-Meyerhof-Parnas 途径，是指在细胞质（胞液）中，1 分子葡萄糖经过 10 步酶促反应裂解为 2 分子丙酮酸，同时净生成 2 分子 ATP 和 2 分子 NADH 的过程；它是几乎所有生物共有的古老葡萄糖分解前期途径，在有氧和无氧条件下均可进行 2012/2017/2021·名解 2012/2024·判断。

请仔细对照上方的 糖酵解十步反应全景图。为什么进化要把一分子六碳葡萄糖变成两分子三碳丙酮酸的过程拆成前 5 步 准备期（耗能阶段，Preparatory / Investment Phase） 和后 5 步 获能期（产能阶段，Payoff / Harvesting Phase）？Vander Heiden 教授带你做了一笔清晰的化学设计账：糖酵解准备期之所以要先倒贴 2 分子 ATP，一是为了给葡萄糖装上带负电的磷酸基团将其“锁”在胞内，二是为了在六碳糖的 C1 和 C6 两端对称装上磷酸基、同时把羰基从 C1 移到 C2，从而允许第 4 步醛缩酶在 C3–C4 之间发生逆羟醛缩合，精准裂解为两个可直接互变的三碳磷酸丙糖！下面我们逐一推演准备期的第 1～5 步：

第 1 步：葡萄糖磷酸化（捕获入胞，第一个不可逆限速点）

α/β-D-葡萄糖（Glc） + ATP ──(己糖激酶（HK）/ 葡萄糖激酶（GK）, Mg²⁺)──► 6-磷酸葡萄糖（G-6-P） + ADP + H⁺

• 热力学偶联推导：如果直接用无机磷酸 P_i 给葡萄糖 C6 位羟基加上磷酸，标准自由能变化是 Δ G'^° = +13.8 kJ/mol（+3.3 kcal/mol），平衡严重偏向左侧，反应根本无法自发进行。因此，细胞将其与 ATP 水解（Δ G'^° = -30.5 kJ/mol，课堂取 -7.5 kcal/mol）偶联，总反应的 Δ G'^° = -16.7 kJ/mol（-4.2 kcal/mol），在生理条件下更是高达 Δ G ≈ -33.5 kJ/mol，成为 糖酵解的第一步不可逆反应，消耗第 1 分子 ATP。
• 为什么要给葡萄糖加磷酸？（高频简答得分点）：葡萄糖经细胞膜上的葡萄糖转运蛋白（GLUT）被动扩散进出细胞；一旦在 C6 位加上磷酸基团变成 G-6-P，糖酵解中间产物全部带强负电荷（生理 pH 下磷酸基带 2 个负电荷），既无法自由穿透疏水的细胞膜脂双层从而将葡萄糖牢牢“锁”在细胞内，又能作为酶活性中心的专属识别基团并降低活化能 2012·单选。
• 催化机制与辅因子：由 己糖激酶（Hexokinase, HK）（肝脏中为同工酶 IV 型 葡萄糖激酶 Glucokinase, GK）催化。注意：所有激酶的真正底物都不是游离的 ATP，而是 Mg²⁺-ATP^2- 复合物（Mg²⁺ 屏蔽三磷酸链上的负电荷排斥，便于葡萄糖 C6-OH 亲核进攻 γ-磷原子）；且己糖激酶是 诱导契合（Induced fit） 模型的经典范例——结合葡萄糖后酶的两个结构域像蟹钳一样闭合，把水分子排斥在活性中心之外，从而防止 ATP 被水分子直接错误水解！

第 2 步：6-磷酸葡萄糖异构化为 6-磷酸果糖（醛糖变酮糖的化学玄机）

6-磷酸葡萄糖（G-6-P，六元吡喃醛糖） ⇌(磷酸己糖异构酶（PGI）) 6-磷酸果糖（F-6-P，五元呋喃酮糖）

• 有机化学机制：由 磷酸己糖异构酶（Phosphoglucose isomerase, PGI，又称磷酸葡糖异构酶） 催化，Δ G'^° = +1.67 kJ/mol，接近平衡、完全可逆。酶先催化环状 G-6-P 开环，利用活性部位的碱性氨基酸残基（Lys/His）作为广义酸碱催化剂，夺取 C2 上的质子形成 顺式烯二醇中间物（cis-enediolate intermediate），随后质子加到 C1 上，将 C1 醛基转化为 C2 酮基，最后闭环成五元呋喃环的 F-6-P。
• 为什么非要把醛糖变成酮糖不可？（MIT 7.05 核心原理）：如果不做这一步异构，直接往下走会遇到两个死胡同！第一，G-6-P 的 C1 是半缩醛羰基碳，无法像醇羟基那样直接被激酶磷酸化，只有异构为 F-6-P 后，C1 才变成自由的伯醇羟基（-CH_2OH）供下一步 PFK-1 装上第二个磷酸；第二，有机化学中的逆羟醛缩合（Retro-aldol cleavage）必须在羰基的 α--β 键与 β--γ 键中的 C_α--C_β 处断裂——当羰基位于 C2 时，C3 是 α-碳、C4 是 β-碳，断裂点正好落在正中间的 C3–C4 键（裂成两个三碳糖）；如果羰基留在 C1，断裂点就会变成 C2–C3 键（裂成二碳 + 四碳），对称拆解就彻底泡汤了！

第 3 步：6-磷酸果糖再磷酸化（真正的承诺步，最关键限速酶）

6-磷酸果糖（F-6-P） + ATP ──(磷酸果糖激酶-1（PFK-1）, Mg²⁺)──► 果糖-1,6-二磷酸（F-1,6-BP） + ADP + H⁺

• 反应特征：由四聚体别构酶 磷酸果糖激酶-1（Phosphofructokinase-1, PFK-1） 催化，Δ G'^° = -14.2 kJ/mol（-3.4 kcal/mol），消耗第 2 分子 ATP，是糖酵解的第二步不可逆反应。
• 为什么 PFK-1 才是整个糖酵解真正的“总阀门”？：糖酵解的速度主要取决于磷酸果糖激酶-1（PFK-1）的活性，它是糖酵解最关键的限速酶（承诺步，Committed step） 2009/2015/2024·单选 2011/2018·填空。为什么第 1 步己糖激酶不是限速酶？因为其产物 G-6-P 是一个多岔路口的中心枢纽（既可走糖酵解，又可去合成糖原/淀粉，还可走戊糖磷酸途径 PPP）；而一旦跨过 PFK-1 生成果糖-1,6-二磷酸（F-1,6-BP），这 6 个碳就别无选择，被 100% 锁定在糖酵解跑道上了！
• 命名预告：之所以叫 PFK-1，是因为它的磷酸基团加在果糖的 C1 位（产物为 F-1,6-BP）；我们在 2.5 节还会遇到一个专门把磷酸加在 C2 位 生成强效调节信号 果糖-2,6-二磷酸（F-2,6-BP） 的调节酶——PFK-2，千万不可混淆！

第 4 步：果糖-1,6-二磷酸裂解为两分子磷酸丙糖（六碳变双三碳）

果糖-1,6-二磷酸（F-1,6-BP） ⇌(醛缩酶（Aldolase）) 磷酸二羟丙酮（DHAP） + 3-磷酸甘油醛（G3P / GAP）

• 有机化学机制（逆羟醛缩合）：由 果糖-1,6-二磷酸醛缩酶（Aldolase） 催化开链形式的 F-1,6-BP 在 C3 与 C4 之间 发生 C–C 键断裂（逆向 Aldol 反应），一步同时生成上方的三碳酮糖 磷酸二羟丙酮（DHAP，来自葡萄糖 C1–C3） 和下方的三碳醛糖 3-磷酸甘油醛（G3P 或 GAP，来自葡萄糖 C4–C6）。
• Ⅰ型醛缩酶（存在于动物和植物中）无需金属离子，利用活性中心的赖氨酸（Lys）ε-NH₂ 与底物 C2 羰基缩合形成质子化席夫碱（Schiff base / 亚胺正离子），充当强力“电子阱（electron sink）”稳定 C3 碳负离子以促进 C3–C4 键断裂；Ⅱ型醛缩酶（存在于细菌、酵母等微生物中）不形成席夫碱，而是依靠活性中心的二价金属离子（Zn²⁺）极化 C2 羰基氧来稳定烯醇负离子中间物。
• 热力学奇观（为什么标准态高度吸能却能正向流畅进行？）：注意看这步的 Δ G'^° = +23.8 kJ/mol（+5.7 kcal/mol）！在标准状态（各反应物与产物浓度均为 1 mol/L）下，平衡极度偏向逆反应（羟醛缩合，这也是为什么这颗酶被命名为“醛缩酶”）。但在活细胞内，一方面产物 DHAP 和 G3P 的实际稳态浓度极低（10^-4 mol/L 量级），一分子底物裂成两分子产物的浓度比项 ([DHAP][G3P])/([F-1,6-BP]) 远小于 1，使得 RT ln Q 为很大的负数；另一方面下游反应不断抽走 G3P，硬生生把胞内实际 Δ G 拉到了 ≈ -1.3 kJ/mol 左右，使反应在生理条件下完全可逆且顺利向右推进！

第 5 步：磷酸丙糖的同分异构化（合二为一，催化完美的扩散限制酶）

磷酸二羟丙酮（DHAP） ⇌(磷酸丙糖异构酶（TPI）) 3-磷酸甘油醛（G3P / GAP）

• 反应机制与汇流逻辑：在醛缩酶裂解产生的两个三碳糖中，只有 3-磷酸甘油醛（G3P）能直接作为后续获能期反应的底物！为了让上方的 DHAP 不被浪费，磷酸丙糖异构酶（Triose phosphate isomerase, TPI） 利用活性中心的 Glu（广义碱）和 His（广义酸），同样经过顺式烯二醇中间体，将酮糖 DHAP 异构化为醛糖 G3P。
• 热力学拉动与“进化完美酶”：该反应的 Δ G'^° = +7.5 kJ/mol（+1.8 kcal/mol），平衡常数 K'_eq ≈ 0.047，意味着平衡时超过 95% 的磷酸丙糖都以 DHAP 形式存在。但正因为下游第 6 步（GAPDH）和第 7 步（PGK）不断消耗 G3P，通过质量作用定律源源不断地将 DHAP 全部抽干转化为 G3P。至此，1 分子六碳葡萄糖经过准备期前 5 步反应，共消耗 2 分子 ATP，100% 汇流转化为 2 分子 3-磷酸甘油醛（G3P）；从第 6 步开始，所有化学计量系数（消耗的 P_i、NAD⁺ 和生成的 NADH、ATP）全部要乘以 2！
• 此外，请记住两个重要考点：① 磷酸丙糖异构酶（TPI）是生物界罕见的“催化完美酶（catalytically perfect enzyme）”，其 k_cat/K_m ≈ 2 × 10⁸ M^-1s^-1 已达到溶液中底物扩散碰撞的物理极限（受扩散限制），任何突变或加速措施都无法再提高其催化速率；其活性中心的柔性环在结合底物后闭合，能防止烯二醇中间体脱去磷酸生成剧毒的甲基乙二醛（methylglyoxal）。② 磷酸二羟丙酮（DHAP）是连接糖代谢与脂质代谢的关键交叉点：在脂肪细胞和肝脏中，DHAP 可被 3-磷酸甘油脱氢酶还原为 3-磷酸甘油（α-磷酸甘油），作为合成三酰甘油（脂肪）和甘油磷脂的甘油骨架前体 2009/2010/2024·单选。

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三、殊途同归：果糖、半乳糖、甘露糖与甘油如何并入糖酵解主干道？

除了葡萄糖之外，我们日常饮食中摄入的蔗糖（水解为葡萄糖 + 果糖）、乳糖（水解为葡萄糖 + 半乳糖）、多糖与糖蛋白中的甘露糖，以及脂肪水解产生的甘油，都不需要另起炉灶建立十步分解通道，而是通过极其精简的“匝道”直接汇入糖酵解准备期的中间产物 2025·判断 2025·简答：

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🎧 MIT 7.05 课堂原声英文实录对照（本节关联 2 讲 · 完整逐段实录）点击展开 / 收起 ▾

🎬 MIT 7.05 Lec 20 · Bioenergetics & Glycolysis I (Prof. Vander Heiden)⏱ ~50:00

~00:00MITOCW | 20. Bioenergetics/Intro Pathways/Glycolysis I PROFESSOR:

~00:02Today we're going to discuss bioenergetics, continue that discussion and use that as a transition into also beginning to see how that applies to understanding glycolysis, the pathway that allows glucose breakdown. So just as a reminder of a couple of the points that we discussed last time, so last time we discussed what makes reactions favorable. And so remember, you could have any reaction, A to B-- A to B could be a single step. A to B could be an entire pathway, all steps in the pathway, that whether or not that reaction or pathway happens is determined by thermodynamics. It's not determined by enzymes. Enzymes are important, of course, to control the rate of reactions so they may allow things to happen that otherwise wouldn't happen. But whether the reaction happens or not is determined by free energy. And so free energy, delta G, remember we discussed is the following relationship. So it's related to this constant delta G0 prime, which is related to the equilibrium constant for any pair of products and reactants as well as this formula rt times the log of the products over the reactants. And so remember this means that whether a reaction happens depends on the equilibrium constant, but it also depends on the actual conditions present. That is how much substrate and how much product is there. And so what this means is that biology can come up with ways by keeping the product concentrations low to do things that may move in the opposite direction of what you would predict from equilibrium. And also by coupling these less favorable reactions to more favorable reactions, you can also allow biology to do unfavorable stuff. And this is, of course, where the role of ATP comes in. And we discussed this at the end of last time as well. Of course, what's important then becomes the ratio of ATP to ADP. And to illustrate that, we use this example. We use the-- remember ATP hydrolysis has a delta G0 prime of minus 7.5 kcals per mole. That, of course, tells you this is about the equilibrium constant. That tells us the equilibrium lies to the right. We can couple it to this first reaction in glycolysis, which traps glucose in the cell by adding a phosphate group. Delta G0 prime for this is positive 3.3 kcals per mole. This positive number tells you that the equilibrium lies towards the left. But when we couple these two reactions together, we add these two numbers together, and now we get a delta G0 prime of minus 4.2 kcals per mole, which are the coupled reaction. The equilibrium lies now again to the right. And that's how we can use ATP hydrolysis in order to carry out this unfavorable process, trapping the glucose in the cell.

~02:38Now how much glucose will be trapped? Well, of course, that will come from this reaction up here. And that's because whether or not a reaction occurs or not of course depends not on the equilibrium constant but on the actual conditions and the delta G of that reaction.

~02:54And so we can just plug that in for this system. And so we see delta G0 prime minus 4.2 kcals per mole. That says equilibrium lies to the right. But how far really depends on the conditions. And that's defined by this ratio of products over reactants. And you can imagine that this term here, as long as it's less than 4.2, you will favor glucose trapping inside the cell, whereas, of course, if it's greater than 4.2, then you'd favor-- the opposite direction would be favorable. Now this is really critical because this really explains how it is that ATP actually works to help cells do various reactions. And so if an ATP-ADP ratio exists such that it can make delta G less than 0 for the reaction that it's coupled to, that is how it provides the energy that then allows unfavorable reactions to occur. And so please do not make the common mistake that many, many biologists make when they state, oh, ATP equals energy. There's a lot of ATP. The cell must have a lot of energy. Remember it's the ratio of ATP to ADP that provides the energy. And so that means ATP can be sky high, but if ADP is equally high, there's no energy there. Consequently, ATP can be very low. But if ADP is extremely low, now you still have a high ATP to ADP ratio. And that is what provides the energy. And that's because this occurs regardless of concentration. It's because it's this ratio of ATP to ADP that ultimately says how much energy is there for the ATP to drive a reaction. In other words, delta G, or whatever the reaction is coupled to, is going to be proportional to the ratio of ADP to ATP. Great. Now how much ATP do you need then? What ratio do you need to drive a reaction? Well, it's hard to say that in absolute terms because it's going to be defined by how that ratio is coupled to the ratio of whatever other reactants and products. It also tells you, remember, as we talked about before, we can switch the directions of these reactions and we just flip the signs. The directions here are arbitrary. And so that means it also tells us how much energy we need to put in if we're going to charge up an ATP-ADP ratio. In other words, if we want to synthesize ATP, that is take ADP plus phosphate and make an ATP, well, what's this? What we just-- the exact opposite sign of that delta G0 prime is going to now equal plus 7.5 kcals per mole. And so that means that the equilibrium of this reaction is going to lie to the left just as that one lied to the right. And it's going to lie to the left by that much. And if we want to know how much energy it then takes to make ATP, well, obviously, it's not going to happen because equilibrium favors this direction. It's not spontaneous in that direction. But at various conditions, we can know by plugging in our formula delta G equals delta G0 prime plus 7.5 kcals per mole plus RT times the log of ATP. So if I have my ADP-- I'm sorry-- my ATP concentration sufficiently low relative to my ADP concentration, I can still make this reaction happen. Now that's not going to give you an ATP-ADP ratio that is actually consistent with being able to do anything else into the cell. And so but what it says is that if I am able to couple energy from nutrient metabolism to this reaction and overcome this positive 7.5 kcals per mole, that basically will allow me to now synthesize ATP and maintain an ATP-ADP ratio in the cell that allows cells to carry out other functions by the same rules that we talked about that would be unfavorable. Now there's lots of implications here for metabolism, because it says how much input you need to synthesize ATP. How much energy you need to synthesize ATP is going to depend on how charged this ratio is. And how useful that ratio is to catalyze other reactions is also going to depend on how charged that ratio is.

~06:50And in essence, this is why we cannot store energy as ATP. This is why we didn't eat ATP for breakfast and instead ate cereal that has a bunch of glucose in it, because we can use that glucose to get ATP but the ATP itself is not something that we really can burn to get energy in a sustained way. In other words, the delta G of ATP synthesis, making ATP is going to be proportional to the log of the ratio of ATP over ADP for all the reasons I described. The delta G of using ATP, ATP hydrolysis, using ATP to drive reactions is also proportional to RT times the log, in this case, of the ADP to ATP ratio. And so because you need to consume this ratio to get energy, and you need to put in energy to create this ratio, you are constant-- and how much is an exponential property-- you are constantly having to generate ATP in a way that allows you to then have a ratio that can then be consumed to fuel other reactions. And that's what's shown here on the slide. So I just graphed here what the ATP-ADP ratio is relative to a theoretical delta G that's shown here as a linear scale, here on a log scale, that basically there's this logarithmic relationship and so you need an exponential increase in energy input to drive this ratio up higher in order to, quote unquote, "store" more energy that can be used for later. And so this system is not one that you can use as an energy storage system and ultimately explains why cells have to do constant metabolism, have to constantly do some sort of metabolism to keep this ATP-ADP ratio charged for it to be in a useful range to have an ATP-ADP ratio that can be coupled to drive otherwise unfavorable reactions in our cells. So to say this another way, our cells need to constantly catabolize nutrients, burn glucose, burn some other fuel. They have to do this because they have to keep that ratio charged in a range where that can then support other cell functions, allow them to fight entropy and maintain order in biology. We alluded last time if you stop making ATP, well, what's going to happen? Well, that ATP-ADP ratio the cell has is going to fuel whatever reactions it can do until suddenly that ratio falls below the point where it's useful to drive those unfavorable reactions. In fact, if you stop ATP production, you will consume that ATP-ADP ratio in seconds. And this is why if you poison ATP production, cells die very quickly. Remember last time we mentioned you have a heart attack, blood flow stops going to a tissue. No more nutrients and oxygen. Cells die quickly. Cyanide poisoning does the same thing. Cells die quickly if you stop their ability to maintain this ATP-ADP ratio in the right range. And so remember the concentration, the absolute concentration of ATP for these reasons is not a good indicator of cell energy. The correct indicator of cell energy is the ratio of ATP to ADP. This term is sometimes referred to as the energy charge. And so that can be the ATP to ADP ratio, or oftentimes more correctly is the ATP to AMP ratio. Why the ATP to AMP ratio? Well, that's because once you understand this, you can now understand that cells have a failsafe mechanism to protect themselves if this ratio of ATP to ADP becomes too low. What is that mechanism? Well, you can carry out this reaction. Take two ATP molecules and turn them into transfer a phosphate to make an ATP plus an AMP. Why is that a failsafe mechanism? Well, if they care about this, if the ATP-ADP ratio is what maintains your ability to drive these thermodynamically unfavorable reactions, if I sacrifice the denominator by removing an ATP and get an ATP out of it, I've now at least temporarily fixed that ratio. Now this is something that obviously cannot go on forever.

~10:36But in the short term, it does allow cells to protect that ratio. And in fact, what cells actually defend is this ATPAMP ratio. And it's very, very difficult to change the ATP-AMP ratio in a cell and actually have it live. So it's really this ATP-AMP ratio that is most formally related to what would be called energy charge. But this is really why this is important is for all the reasons that I just described. All right. So hopefully, it's clear to you now why cells have to keep ATP-ADP ratio in the right range, why this is useful for them to now carry out all of these unfavorable reactions-- fight entropy, stave off death. And they can even have a failsafe here and sacrifice ADP as a way to try to maintain that as a very last resort. It also says why cells have to always do catabolism, because you always need the energy input to keep that ratio in the right range. However, you might imagine that, in real life, the messiness of biology, that there's lots of situations where the demand for ATP will suddenly increase. Well, of course, maybe you can just metabolize more sugar, do more nutrient catabolism to try to keep that ratio high. But that takes time. And so you need ways for cells to quickly respond to then allow them to mobilize all of this extra thing to-- extra fuel to do that. For example, we're walking around our ancestors on the plains somewhere and a lion appears. We don't have time to mobilize a bunch of new fuel. We have to be able to run away really quickly or the lion's going to eat us. And so cells have systems to suddenly respond to I need a lot more ATP demand to run away by having ways that one can buffer this ATP-ADP ratio, protect it in the short term, and allow cells time to ramp up nutrient metabolism in order to keep this high. All right. Well, if we wanted to create such a buffer system, how would we actually do it? Well, obviously, we have to follow all the rules of thermodynamics that we discussed. And we have to do so in a way that actually illustrates how it is that very well how metabolism works. In other words, if suddenly the ATP-ADP ratio drops, if we have a sudden drop in ATP-ADP ratio because we have increased demand, needing to run away from a lion, how could we actually create a buffer system that protects this? Well, to do it, we would need to couple ATP-ADP to some other product reactant pair, let's call them x and y, such that the ratio of y to x is coupled to the ratio of ATP to ADP. And you want it coupled in such a way that if that ratio goes down, this ratio can now protect ATP, basically favor ATP to ADP synthesis. However, when nutrients are plentiful and this ratio is really high, it then in turn can charge up this ratio so that it can buffer it later when the ATP-ADP ratio starts to fall. Now the most famous example of this actually comes from muscle physiology. And it's best described in terms of muscles and how they quickly respond to increased demand, like that running away from a lion. However, it in fact applies to many, many cells, including most non-muscle cells in animals.

~13:47All right. And I should say that it turns out that if you turn off ATP synthesis in a muscle, people have estimated that your ATP-ADP ratio will fall into a range where it is no longer useful to do anything within a matter of seconds, some claim even less than a second. And so this system ends up being very important.

~14:08Now the system that muscle uses, and many other cells do as well, is something called the creatine/creatine phosphate system. Now if you guys are weightlifters or bodybuilders out there, you probably have heard of creatinine because it's a very popular supplement that is taken for weightlifting, something I obviously don't do a lot of. But that creatine/creatine phosphate is out there. I'll describe to you what it actually does for muscle cells. And you can decide for yourself if this is actually useful as a energy or weightlifting supplement. So what is creatine/creatine phosphate? What does it look like? Well, it's basically looks like this. By the way, I will use this abbreviation often throughout this course, just a shorthand to draw a lot of these structures. So this here would be a carboxylic acid group drawn like this or like that, drawn to the carboxylic acid group just so you understand my shorthand. OK. So this here is the molecule creatine. If I put a phosphate here, this now becomes creatine phosphate. Again, just to remind you, this P with a circle over it is a phosphate group. I'll draw this particular phosphate group because it's a nitrogen-phosphate bond, a phosphoramidate bond. This is actually a bond that is more labile than the PO bonds that we're used to seeing in the phosphodiesters like ATP, et cetera. All right. So that's creatine and creatine phosphate. And if we draw out the reaction, creatine phosphate goes to creatine plus inorganic phosphate, so hydrolysis of creatine phosphate. This has a delta G0 prime of minus 10 kcals per mole. What does that mean? It's negative. Tells us about the equilibrium constant. Equilibrium lies to the right. Remember ATP to ADP hydrolysis shown over there, that hydrolysis has a delta G0 prime of minus 7.5 kcals per mole. Equilibrium also lies to the right. This number is more negative. This equilibrium lies further to the right than ATP hydrolysis does. So now what happens if we couple this reaction to the ATP hydrolysis reaction? So now we can do ATP plus creatine goes to creatine phosphate plus ADP. So we've coupled those two reactions together. So delta G0 prime, ATP to ADP is minus 7.5. Creatine phosphate to creatine in that direction is negative 10. In this direction, it would be positive 10. And so as I've drawn the reaction, this delta G0 prime will be plus 2.5 kcals per mole. 10 minus 7.5 plus 2.5, which tells us then that this equilibrium now lies to the left. However, which direction this reaction actually goes is not determined only by the equilibrium. It's really determined by delta G. So we can calculate that. So delta G equals delta G0 prime plus RT log of, as we've drawn it, creatine phosphate over creatine and ADP over ATP. So you can now imagine that this term here is the sum of those two. So it's plus 2.5. And so if this term is less than 2.5, what does that mean? Well, if it's-- I'm sorry. Less than negative 2.5, then delta G will be negative and you will favor creatine phosphate production. If this term is greater than negative 2.5, then delta G will be positive and you will favor ATP production. Why is that important? Well, if you think about the conditions, how you could make this term less than 2.5 or less than negative 2.5 or greater than negative 2.5, basically, what's going to happen is that if the ATP-ADP ratio is high, lots of energy around, no problems charging up, lots of things to use, charge up this ATP/ADP to a very high ratio, well, now you will favor this direction, creatine phosphate synthesis.

~17:33However, if ATP/ADP begins to fall and is lower, well, now this term is going to flip and you'll now favor ATP production. And so basically, by charging up this creatine/creatine phosphate ratio, it now creates a situation where it can buffer that ATP/ADP ratio so that when times are good, you charge up the buffer. When times are bad, you then consume the buffer in a way that allows you to protect your ATP/ADP ratio for a short time. And this is illustrated very nicely here in this slide that basically shows what happens in muscle physiology. And so this is just a theoretical curve that if you suddenly need to contract your muscle and you stop ATP production, well, ATP levels will fall. That ATP/ADP ratio more correctly will fall rapidly within the cell. Creatine phosphate will then now kick in to protect that ATP/ADP ratio as you can ramp up either anaerobic or aerobic metabolism to now buffer that ATP/ADP ratio and keep that muscle functioning. Now I want to make one point because oftentimes biochemistry textbooks will refer to things like ATP or creatine phosphate as, quote unquote, "high energy compounds." And of course, I guess they are high energy because they have these labile phosphate-nitrogen or phosphate-oxygen bonds. And because they're labile, hydrolysis is favorable. And to a certain extent, it's that hydrolysis that is providing energy. But remember it's not the metabolite or the bond in isolation that's really providing the energy. It's these ratios of ATP to ADP, creatine phosphate to creatine, that allow you to couple, for all the reasons we've spent now a lecture and a half discussing, two other reactions that really provides biological energy. All right. Now we're ready to discuss how it is that we can use glucose oxidation, nutrient catabolism, to keep ATP/ADP high, that is in a useful range, where it can allow cells to do otherwise thermodynamically unfavorable things. So how could we make this happen? Well, just like this example with creatine and creatine phosphate, if we want to use nutrient catabolism to do the same thing, we need to couple reactions of glucose oxidation, nutrient catabolism, that will allow us to synthesize ATP, ADP to ATP, and have that synthesis be favorable despite the high ATP/ADP ratio in cells. So let's think about it. So what is this? Nutrient catabolism. What did we talk about last time? Oxidizing glucose to carbon and CO2. So here's our reaction for that. So here's carbohydrate, glucose CH2O6 plus six O2 goes to six CO2 plus six H2O. That's the chemical formula for burning wood. So delta G0 prime for this is minus 686 kcals per mole. Quite a negative number. Equilibrium greatly favors the completely burned state, oxidized state, of that carbohydrate. And so you can imagine that this equilibrium is so favorable to the right, if we couple this reaction to ATP synthesis, you're clearly going to have plenty of energy there in order to maintain that ATP/ADP ratio in the right range to be compatible with cells using it to do other things. All right. So how do we actually do that? Well, if we do it, we have to follow the same rules of thermodynamics and do the same things that we did before. Basically, we have to do this reaction in a way that we can draw out a similar type of equation as we did for creatine and creatine phosphate. And at the highest level, that is how nutrient catabolism will allow us to keep ATP/ADP ratio high even as ATP hydrolysis is constantly fueling all these other unfavorable processes in the cell.

~20:55Now if we did this all in one step, our burning wood example, lots of heat is generated. That's too much energy. Not so good for biology. But rather what biology does, as we alluded to last time, is that because what we talked about in the beginning, that all of the things that we discussed are true for individual reactions or many, many reactions, basically, if we turn A into B and we do it in one step, like burning wood, or we do it in 500 steps, the thermodynamic consequences are exactly the same. And so what we need to do is do stepwise catabolism of glucose and take advantage of the fact that that delta G change is the same whether it's one step or many steps and in that stepwise fashion build in reactions where we create intermediate chemistries that allow us to do things like we did with creatine and creatine phosphate such that we can favor ATP synthesis when ATP/ADP ratio is in the physiological range. And in essence, this is the magic of how biology does this and basically couples nutrient catabolism in order to provide energy. And so the pathway that we're going to discuss today and in the next lecture is something called glycolysis, which simply put is glucose lysis. It's the stepwise chemical oxidation of glucose, which we know from our burning wood example is favorable. And we're going to use this pathway that basically will be a pathway to allow the stepwise lysis of glucose, a favorable reaction, to individual steps that set it up in a way such that ADP to ATP synthesis is favored despite a physiological ATP/ADP ratio. Now I want to point out that this really illustrates, back to that high energy compound thing, that ATP is not the only energy currency in the cell. In fact, glucose is much more energy for a cell than ATP. You get much more energy released from burning wood than you get from hydrolyzing ATP. And in essence, this is why we use glucose-- why nature has used glucose, and other molecules like it, as an energy storage device, because here is a relatively stable molecule, can sit around for a long time, and you can burn it when you need it to keep your ATP-ADP ratio high. All right. So, at the level of reactions and pathways now, how can we use glucose oxidation and couple it to drive ATP synthesis? Well, hopefully it's very clear that we have to basically set up a situation like this creatinecreatine-phosphate system that we already discussed. And so I want to start by discussing, what are the reactions in glycolysis that ultimately behave like this and allow this favorable ADP-to-ATP synthesis? And then we will go ahead and consider how we can make those reactions work in the context of an entire pathway that allows glucose oxidation. All right. Now, like creatine, phosphate, and creatine, both of the steps in glycolysis that favor ATP synthesis involve intermediates where you have phosphate on some molecule, plus ADP goes to ATP plus the phosphate coming off that molecule. Now, if we go to our creatine-creatine-phosphate system, obviously this equilibrium needs to lie to the right. And so the first one of these is as follows. It involves this molecule, phosphoenolpyruvate, also referred to as PEP. You will see throughout the course that we love to give these little acronyms to our metabolites because it's hard to constantly say and write out phosphoenolpyruvate. Much easier to say PEP. So phosphoenolpyruvate plus ADP goes to ATP. Plus, if I take the phosphate off of there, I get this molecule. This molecule is enolpyruvate. Enolpyruvate can be rearranged as follows. To make this molecule a ketone pyruvate.

~24:24You'll note-- hopefully remember from your organic chemistry classes that ketones like pyruvate can exist in two forms. They can exist in the ketone form or the enol form, rearranging via the chemistry that I've shown. The keto form is greatly favored over the enol form. However, by having a phosphate trap pyruvate the enol form-phosphoenolpyruvate, when you remove the phosphate and it's wanting to exist in the keto form, greatly enhances the energetics of this-- basically makes the reaction, the equilibrium want to lie very far to the right. And by the way of chemical tricks like this and others like it, you will see used over and over in biology. In fact, this is illustrated, if we asked what's delta G0 prime for this reaction, it's minus 15 kcals per mole. That tells us that the equilibrium of this reaction lies to the right. In fact, it lies far to the right, and in fact, this is-- you may remember ATP hydrolysis is negative 7.5 kcals per mole. So lies twice as far to the right as ATP hydrolysis does. And in fact, this minus 15 kcals per mole is the most favorable, at least in terms of individual reactions that at least I know of in metabolism. All right. Well, if this equilibrium for this is PEP to pyruvate, this here is delta G0 prime for ATP-ADP. So if we want to know what's delta G0 prime for the entire reaction, well, if PEP to pyruvate is minus 15, ATP to ADP is minus 7.5. Well here, we're going in the opposite direction, so it'd be plus 7.5. So it's minus 15 plus 7.5, means that for the entire reaction, this is minus 7.5 kcals per mole.

~26:00Means that this is favorable, equilibrium lies to the right. If we drew out-- I'm not going to do it again because I've done it a lot, but if we redrew out this equation, replacing pyruvate and PEP in the correct places, there for creatine, phosphate, and creatine, you would see that there's lots of conditions within cells that will favor ATP synthesis even when ATP-ADP ratio is high. All right. This reaction is catalyzed by an enzyme called pyruvate kinase. And pyruvate kinase is a classic example of a, quote-unquote, "irreversible reaction." However, I want to point out that the enzyme pyruvate kinase is actually named for the reverse reaction that's supposedly irreversible. Pyruvate kinase. If you take pyruvate and you act on the kinase as you learn from Professor Yaffe, that phosphorylates something. You phosphorylate pyruvate, you get phosphoenolpyruvate pyruvate kinase. And so remember that absolutely no reaction is reversible. It's only reversed-- what they mean by irreversible when they say pyruvate kinase step in glycolysis is irreversible, is that it is effectively irreversible under the conditions that are compatible with life that exist in cells. Of course, if you have very low ADP and very high ATP, you can come up with a way to net synthesize PEP. You just can't drive the reverse reaction under the ATP-ADP and the PEP pyruvate ratios that physiologically exist in cells. However, this is set up in a way such that you can favor ADP-to-ATP synthesis by converting PEP to pyruvate in a pathway, as we will see happens in glycolysis. All right. Now, the second reaction that I want to-- that allows ATP synthesis in glycolysis is catalyzed by an enzyme called phosphoglycerate kinase. And this carries out the following reaction. OK. So this molecule is 1,3-Bisphosphoglycerate, abbreviated 1,3-BPG. And basically the phosphate is transferred from here to ADP, which gives you this molecule, 3-phosphoglycerate or 3-PG.

~27:44So this reaction, the delta G0 prime for the entire coupled reaction, both steps of the reaction, is minus 4.5 kcals per mole. Tells you about the equilibrium constant. Equilibrium lies to the right, doesn't lie as far to the right as the PEP plus ADP goes to ATP plus pyruvate reaction, but nonetheless, is one where there's lots of conditions that are favorable in cells to maintain a high ATP-ADP ratio, although we will see later that this reaction actually runs quite close to equilibrium in cells, so there's actually conditions where it can go in the opposite direction as well, and we'll get to that later in the course. I want to point out one thing about this, one thing that makes this reaction favorable, as you notice here that this is a phosphate on an acid group, a so-called acid anhydride. And this is a pretty good phosphate donor. And so you want to lose that phosphate, which is one of the reasons why this reaction is favorable for ATP synthesis even at high ATP-ADP ratios. All right. So these are the two reactions in glycolysis that can favor ATP synthesis at a high ATP-ADP ratio just like creatine and creatine phosphate. And so hopefully now it's become apparent to you that if during our pathway of glucose catabolism we can build a pathway that couples what we already know is favorable, glucose oxidation, to generate these intermediates, PEP and 1,3-Bisphosphoglycerate, lacerate we can then build a pathway where those reactions can make synthesis of ATP favorable even though there's a high ATP-ADP ratio in cells, and in fact, maintain that high ATP-ADP ratio in cells. And in essence, this is the logic or the goal, if you will, of glycolysis and why it's important to provide energy to cells. OK. All right. Now, if we're going to make a pathway where glucose oxidation is coupled to making PEP and 1,3Bisphosphoglycerate, it's obvious we need to add phosphate to the system. There's no phosphate molecule on glucose. Now already mentioned earlier-- and I just erased it-- that to trap glucose in the cell, the first step in glycolysis, you add phosphate. But that phosphate donor is ATP. And so ATP being the phosphate donor doesn't help us here. If we're going to use phosphate transfer here, we need to have phosphate added into the system if this is going to work. We can't have ATP as our phosphate donor. You also saw lots of reactions from Professor Yaffe that involved phosphate transfer reactions. But again, ATP-- or GTP is the phosphate donor. And so for this system to work, we need a way to get inorganic phosphate onto a carbohydrate or we can't make things like PEP or 1,3-Bisphosphoglycerate that will allow ATP synthesis. Now we already saw earlier that additional phosphate to glucose is not favored. Remember, delta G0 prime of phosphorylating glucose was plus 3.3 kcals per mole. And it turns out that says equilibrium lies to the wrong side, so we can't net do that without ATP or energy input. And it turns out this is true for other sugars as well. And so you need energy input to add phosphate to the sugars. Plenty of energy input from glucose oxidation, but we need a reaction where phosphate addition is coupled to another reaction that makes it favorable, and we have to do this without making ATP. And again, it illustrates, again, the biological energy is not just about ATP. At a deeper level It's something different. And so to really discuss this and understand it, I need to introduce two concepts that are going to come up again over and over again in this class and across metabolism. The first of these is a very high-level bioenergetic concept, although it's really an energetic concept, and it comes back to what we already talked about.

~31:19So, we know burning wood is favorable. The CN H2O N plus oxygen going to CO2 plus water. That's burning wood, lots of energy released. Drew it up there for a single-glucose molecule and what the energy release the delta G0 prime is for that reaction. Now I want to point out chemically what is going on in this reaction. Chemically what is happening for this to occur is that electrons from the carbon are being moved to the oxygen. You are changing the oxidation state of carbon and of oxygen in order to carry out this reaction. Oxygen is a better electron acceptor than carbon, and so transferring those electrons from the carbon to the oxygen is favorable. That is, in essence, what is going on in this very favorable reaction to burn wood. Let me illustrate that a bit more explicitly here. So the carbon-- most of the carbons in carbohydrates are alcohols. So here, you can call this our carbohydrate, with the exception of the ketone or the aldehyde if it's a ketose or an aldose. All the other carbons in the sugar are alcohols. All the other bonds there. And so if we oxidize that carbon-- that is, if we remove electrons from the carbon, how can we do that? So this here generates this H- hydride ion. This is, in essence, two electrons that we are removing from the carbon. That gives us a ketone. So if we oxidize the alcohol and we remove two electrons, we get the ketone. We could oxidize this ketone further. That will give us the carboxylic acid. If we oxidize it even further, now we get CO2. So, moving in this direction, this is oxidation. Transferring electrons from the carbon to the oxygen to go in this oxidation direction, that is glucose oxidation is what's happening when we're burning wood. It's what happens when we burn glucose. Energy is released. This is largely what catabolism is about in biological systems. Now then reasons, then, that if we're going to move in the opposite direction, so-called reduction, that is going in this direction. Reduction. That's largely energy storage or anabolism in metabolism. We are storing energy that we can burn later. And of course, what's the more extreme version of this? Well, what if we add two more electrons to the carbon going in this direction? Well, what do we end up with? Well, now we end up with the saturated carbon. more reduced than the alcohol is the fully saturated carbon. You will see later that this is what fat is. You saw that in some of the earlier lectures. It's also what gasoline is. What's gasoline? It's fully saturated hydrocarbons that we then burn to CO2 and derive energy. Just like burning wood, burning gasoline all in one step, oxidizing the gasoline gives you energy. There's more energy in gasoline. You can release more energy per weight from gasoline, from fully-saturated hydrocarbons than you can from wood. Same thing happening here. What has more calories in your food? Everyone knows fat has more calories per weight than sugar. It's because the more reduced our carbon is, the more energy it stores, and as we oxidize that carbon, that is what releases the energy. And so energy transduction in biological systems-- and actually, in non-biological systems, as I just illustrated with the wood and gasoline example, but definitely in biological systems, is largely about oxidation and reduction reactions, and you are going to see this coming up over and over again.

~34:34Now, based on what I just said, the real magic, if you will, of glycolysis can be further refined to say that what glycolysis does is it couples the favorable oxidation of carbon with reactions that allow phosphate additions and production of intermediates, like PEP and 1,3-Bisphosphoglycerate, where synthesis of ATP is favored despite the high ATP-ADP ratio in cells. If you think about it, that process then allows you to keep that ATP-ADP ratio in the range where it can then be useful for all the reasons we've been talking about now for two lectures. Allows the cell to couple that ATP-ADP ratio, that-- in ATP hydrolysis to otherwise carry out otherwise unfavorable reactions. OK. So, based on that, arguably the key, or at least a very important reaction in glycolysis, is the oxidation step which happens to add the phosphate and also happens to make 1,3-Bisphosphoglycerate, which we saw earlier can be used to drive ATP synthesis. And so that reaction is the following. So this molecule hopefully looks familiar to you from the carbohydrate lectures. This is D-glyceraldehyde 3phosphate. I will abbreviate it frequently is G-3P for glyceraldehyde 3-phosphate, but it's D-glyceraldehyde 3phosphate. Remember, it's a D, sugar, because the OH group is pointing to the right as we drew it this way. It's also an aldose because it's a aldehyde sugar, and it's phosphorylated on the 3 position-- remember the way we numbered sugars was starting at the carbonyl-- 1, 2, 3. This is carbon-3, phosphorylation on the 3 position. Glyceraldehyde 3-phosphate. OK. So, this step generates 1,3-Bisphosphoglycerate. Well, to do this, there's actually two things that happened here. First we had to add the phosphate to this. It says two phosphates on it. bisphosphoglycerate, this has a single phosphate. So inorganic phosphate has to be added to generate that molecule. By the way, adding that phosphate delta G0 prime is positive plus 1.5 kcals per mole. That means even equilibrium, this reaction would lie to the left, of course, because delta G0 prime is positive. However, remember the next step downstream of this is the one shown over here where we do ATP synthesis. That step is very favorable. So this is an example where metabolism has coupled a very favorable step to a step that's less favorable. That can keep the 1,3-BPG levels low and pull the entire system forward, the metabolic trick we discussed in the last lecture. OK. You'll note the other thing that's happening here, other than adding that phosphate group, is that we've changed this aldehyde to a carboxylic acid. That is an oxidation reaction. Ketone to the acid is an oxidation reaction. I will illustrate over here this so you can see why it's an oxidation reaction. So here's our aldehyde. If we take electrons from the carbon and that generates this hydride ion to electrons, what are we left with? Well, we're left with this intermediate that will never exist with a positive charge on the carbon. Water, which, of course, is abundant in biological systems. And then come-- OK. And it basically illustrates that as I go from the aldehyde to the acid, of course, I make a proton and a hydride ion, but really, I have changed the oxidation state of the carbon from the ketone to the acid state. Two electrons are lost there, is shown as that hydride ion. OK. So, what does that mean? Well, that means that we have to maintain that balance in this reaction as well. So water is going to have to come in and a proton will be produced. But the key thing is we also generate two electrons, this hydride ion that have to go somewhere. Now, finding a place for those two electrons to go is what brings up the second high-level concept that I now need to spend some time discussing.

~38:08And this second high-level concept that I need to introduce is the role of cofactors, and cofactors also is effectively the role of vitamins and what role that these things play in metabolism. Most vitamins are used to support metabolic reactions, and we're going to learn all about what many of the vitamins that you guys are familiar with reading the side of your cereal box actually do throughout the course.

~38:31Now cofactors, as I said, often involve vitamins, are molecules that provide useful chemical groups that facilitate the reaction chemistries that are needed in metabolism. So they provide some useful chemical groups to facilitate some of the chemistries that we need to carry out metabolic reactions. They are, in general, in small non-stoichiometric quantities in order to carry out this function. What do I mean by that? Meaning that you can imagine that the cell will convert lots of glyceraldehyde 3-phosphate to 1,3Bisphosphoglycerate, but the enzyme, you don't need one enzyme for every time you convert that reaction. The same enzyme can do it over and over and over again. Well, it turns out, cofactors are much more like enzymes in that respect. They need to be recycled in a way that they can catalyze the turnover of reaction many, many, many times, and it'll be much clearer what I mean by this when we get into some of the details. Now, the vitamin concept comes from the fact that often these cofactors or parts of the cofactors are not made by animals, not made by humans. If they're not made by us, but we need them to do our metabolism, well now we have to get them from the diet, and this is really the concept of vitamins. These are small things that we have to get in quantities from the diet, and it turns out they're very important to help us carry out the metabolism we do. As an aside, the vitamin industry is a very big business. Lots of unfounded claims out there. I'm not going to get into-- that can get very political. I'm just going to say that just know that more is not always better, and the goal for this class is really to understand what some of these vitamins actually do. OK. Now, the relevant cofactor for the reaction that we're talking about is a cofactor called nicotinamide adenine dinucleotide, which is always abbreviated NAD or NAD+. This stands for nicotinamide adenine dinucleotide. You see why we want to abbreviate that NAD. And basically what nicotinamide adenine dinucleotide is is it's a factor that can solve our problem of where to put these electrons in this oxidation reaction. That is, you can accept two electrons from a reaction like this one here and cycle to another form of the cofactor called NADH. All right. What is nicotinamide adenine dinucleotide? Well first, let's draw what it looks like. OK. Sorry it's squeeze down here a little bit. So this here is adenine. This group up here is nicotinamide. So if you look at this, this is a dinucleotide. And so down here, this, of course, is, from here down, is AMP. That phosphate is now over here. This is a separate nucleotide, where rather than having one of the AGCTU bases that you're used to from RNA and DNA, nicotinamide is the base. And so it's this AMP plus this nicotinamide base is a monophosphate, or a nicotinamide adenine dinucleotide. This, the nicotinamide group, is derived from a vitamin called niacin. And so from the side of your cereal box, this is what niacin does. It allows you to generate the nicotinamide group that's part of this cofactor, nicotinamide adenine dinucleotide. Now, nicotinamide adenine dinucleotide is useful because of what that nicotinamide group can do.

~41:36And so I'm not going to draw out the whole molecule, but I'll show you. So NAD+, which is the oxidized form of nicotinamide adenine dinucleotide, is as I drew it up there. So this here would be attached to the rest of the dinucleotide. So this is the oxidized form. I can have-- here's my hydride ion, which, remember, is two electrons. If I carry out that chemistry to add those two electrons to the nicotinamide group, you now end up with the reduced form of nicotinamide adenine dinucleotide NADH. So the difference between NAD+ and NADH, it's the H, but it's really the two electrons that were added in adding that H. So it's oxidized form and reduced form. So remember, I want to stress this again, at least in biology we can't create or destroy matter. You can do that in nuclear physics, but we can't do that in biology. And so that means that if we do an electron transfer like we did in this reaction where we oxidized the aldehyde in glyceraldehyde 3-phosphate to the acid in 1,3Bisphosphoglycerate, those electrons have to be accounted for. And so if one molecule is oxidized, another one has to be reduced. So redox reactions always-- that is, redox, oxidation-reduction reactions, always have to happen in pairs. And so over there we have the aldehyde in glyceraldehyde 3-phosphate is oxidized to form the acid in 1,3-Bisphosphoglycerate. As part of that reaction, we can couple it such that the nicotinamide group, which is oxidized in NAD, can be reduced to the nicotinamide group that is found in NADH. So that means we can draw this in here, is that having that cofactor coupled to this reaction now allows us to fix the redox problem of this reaction. Now this reaction here is catalyzed by an enzyme called glycerol 3-phosphate dehydrogenase. I'll draw that out in the second. Often abbreviated GAPDH, the loading control in all of your western blots, is the enzyme GAPDH, glyceraldehyde phosphate dehydrogenase. That is the enzyme that carries out that reaction. Now notice, the name fits. It's a dehydrogenase, it's removing a hydrogen, removing two electrons as a hydride ion, but it's really-- what's important here is the electrons moving, not the hydrogen itself. So in that way it's a little bit misleading. And you will see that in general, dehydrogenases are classes of enzymes that catalyze oxidation-reduction reactions, and as a result, often involve NAD/NADH as cofactors because they facilitate those electron transfer reactions. OK. So now we're able to describe how GAPDH works in great detail. And so the chemistry of GAPDH works as follows. And so in the active site of GAPDH, there's a cystine residue. That cystine residue has a sulfur shown here. Also bound near the active site is in NAD molecule to catalyze the reaction shown. That NAD has to be in the oxidized state, so it's NAD+. And so glyceraldehyde 3-phosphate binds in the active site. There's glyceraldehyde 3-phosphate. By the way, I will show you how many reactions in metabolism work chemically. Obviously we don't have time in this class, an overview class to go into all the great chemical details of what's going on and exactly how everything works, but I'll at least try to give you a flavor of some of the chemistry that happens throughout the course. All right. So once you have, then-- OK. So now you have this intermediate bound into the enzyme active site. You can now carry out the oxidation of that carbon. So these are two electrons being removed as a hydride ion. They can be transferred to NAD in the active site. That generates NADH by the exact chemistry that I just showed you. OK.

~45:03That NADH now needs to be exchanged out or the enzyme will not be able to catalyze the next series of reactions, and that NADH will then go somewhere else, and of course, has to be cycled back to NAD, but that doesn't happen in this reaction, per se. OK. Next, this here, you'll notice, is that by the way this chemistry worked, we formed this thioester bond in the active site. This is another thing that you will see over and over again in metabolism. This sulfur in the thioester bond is a good leaving group, and so it's really setting up a situation that will now help with the phosphate addition. So here, I'll draw out inorganic phosphate. And that, of course, will give us the identical enzyme active site that we started with plus 1,3-Bisphosphoglycerate. And that 1,3-Bisphosphoglycerate can now be coupled to ATP synthesis to make 3-phosphoglycerate. And so you can see, in essence, what this reaction has done is that it has coupled oxidation of the aldehyde to the acid, the aldehyde in 3-phosphate to the acid in 1,3-Bisphosphoglycerate, which can then do phosphorylation of ADP to make ATP. So in essence, this is oxidative phosphorylation. Now I realize in high school or in other classes, oxidative phosphorylation means something different to you, and of course, we will discuss the process that is called oxidative phosphorylation in great detail later in this course. But I want to point out that the chemistry that's actually happening here in glycolysis is also oxidative phosphorylation. It's basically how you coupled this favorable carbon oxidation that's occurring at this GAPDH step in a way that allows you to synthesize ATP at a high ATP-ADP ratio, which in the end will allow cells to do otherwise unfavorable things. OK. Now we're ready to discuss these reactions, the GAPDH reaction, the pyruvate kinase reaction, the phosphoglycerate kinase reaction, and how they fit into glycolysis as a metabolic pathway in a way that allows cells to use glucose metabolism-- that is, the breakdown of glucose, the burning of glucose as a way to get energy. And that pathway is, of course, glycolysis or glucose lysis that we mentioned earlier. Glycolysis is a very ancient and ubiquitous pathway. It's used by essentially all life on Earth and probably has been for at least 2 to 3 billion years. Glycolysis, I guess, as a pathway was, quote-unquote, "discovered" mid-19th century by Louis Pasteur, and Louis Pasteur, of course, described life, microorganisms as being responsible for the process of fermentation. That is, conversion of glucose into alcohol, something many college students are interested in, as well as other organic acids. And of course, fermentation to make alcohol or to store other types of organic acids has been used in food preservation for centuries, which is really why Pasteur's discoveries were so impactful at the time. Now even though Pasteur discovered this, he didn't really understand chemically how it worked, and it actually took decades to figure that out. And when it was finally figured out and described as the pathway called glycolysis, this was done by two chemists, two chemists in Germany, Embden and Meyerhof. And basically what they did is they pieced together several enzymatic or chemical activities that have been purified from cell lysates mostly from yeast and used those to build a pathway that described chemically how you could start with glucose and chemically break down that glucose in a way that made sense in this fermentation pathway, and that is why glycolysis is sometimes called the Embden-Meyerhof pathway.

~48:20Now, glycolysis does not completely convert glucose to CO2. Instead, what glycolysis does is it converts glucose-so I will draw here in the pyranose form. It converts glucose by a number of steps into two molecules of the organic acid pyruvate, which, of course, was the product of that pyruvate kinase reaction that we showed earlier. Note that the fermentation that Louis Pasteur described was further metabolism of that pyruvate into something else. In the case of yeast, it was ethanol or ethyl alcohol, or something else. The fermentative product in mammals is this organic acid lactate. And what we will see next time is that this fermentation of pyruvate into one of these other molecules ends up being an alternative to the further oxidation of pyruvate to CO2 which requires oxygen, and the reason oxidizing pyruvate to CO2 requires oxygen is because ultimately we have to regenerate that NADH we made in glycolysis to allow that cofactor to cycle and glycolysis to keep moving. Fermentation gives us an alternative way to do this, which we'll discuss in great detail in the next lecture. But first I want to focus on glycolysis and what we can describe next time as really how we can build a pathway to turn glucose into pyruvate. This glycolysis, this Embden-Meyerhof pathway, and some of the details of that pathway are shown here on this slide. And this is where we'll start off the next lecture, discussing how each of these steps in glycolysis function in a way that allow a pathway to generate phosphoenolpyruvate, 1,3-Bisphosphoglycerate, incorporate this GAPDH reaction, and do so in a way that's overall energetically favorable to allow the cell to couple this oxidation of glucose to keeping ATP-ADP ratio in the right range to support other unfavorable functions in the cell. OK, thanks.

🎬 MIT 7.05 Lec 21 · Glycolysis II & Allosteric/Hormonal Regulation⏱ ~50:00

~00:00MITOCW | 21. Glycolysis II/Regulation

~00:01MATTHEW

~00:02OK. Today we will start our discussion of-- with a discussion of glycolysis as a pathway and how we can use

~00:09VANDER

~00:09favorable oxidation of glucose to enable phosphate addition while at the same time producing useful

~00:15HEIDEN:

~00:15intermediates that enable us to do ATP synthase-- ATP synthesis and maintain a high ATP-ADP ratio in cells. Now I drew up here the entire glycolytic pathway, just like you saw at the end of the last lecture, and I want to go through it here step by step. And so the first step in glycolysis, as we already talked about, is we have glucose. This here is, of course, alpha-D-Glucose in the-- I drew it in the alpha form. If the OH was pointing up, that would be the beta form, just some quick review. I'll drop the alpha and beta and the stereochemistry denotion going forward. This glucose molecule, as we discussed previously, is first trapped in cells by being phosphorylated by the enzyme hexokinase, sometimes abbreviated HK, to give glucose 6-phosphate. Not shown here, this glucose 6phosphate then undergoes an isomerase reaction via the open chain form. Again, we discussed this reaction when we discussed sugars. And so this would be catalyzed by glucose phosphate isomerase, and takes it from the aldose to the ketose, and now you have the fructose-- the ketose fructose 6-phosphate shown here in the furanose form. This fructose 6phosphate is then phosphorylated a second time by an enzyme called phosphofructokinase, typically abbreviated PVK. It's phosphorylated on the 1 position to give fructose 1,6-bisphosphate, shown here. That fructose 1,6-bisphosphate can then be cleaved via the enzyme aldolase. Cleavage takes place in the open chain form which I drew up here in the corner. Basically splitting the molecule in half to form two trioses, one of which is the ketose dihydroxyacetone phosphate, basically the top half of the molecule. The other is the bottom half of the molecule, the aldose glyceraldehyde 3-phosphate. These two trioses can be interconverted, the aldose and the ketose via on isomerase. The enzyme that does that is called trioses phosphate isomerase. And moving forward basically takes the dihydroxyacetone phosphate that comes from the top half and can turn all the carbon from fructose 1,6-bisphosphate into two glyceraldehyde 3phosphates. That glyceraldehyde 3-phosphate, as we discussed last time, is the substrate for the GAPDH reaction, the glyceraldehyde 3-phosphate dehydrogenase, GAPDH. which, as we discussed last time, couples oxidation of this aldehyde to this acid with phosphate addition. That oxidation reaction, the electrons have to go somewhere, that's where the cofactor NAD comes in. Picks up two electrons, generates NADH, and in the process, this enzyme generates this molecule, 1,3-bisphosphoglycerate or 1,3-BPG.

~02:41Remember, that 1,3-BPG molecule was one of the useful intermediates whereby we could undergo favorable synthesis of ATP even at the high ATP-ADP ratio found in cells, and that's because we can transfer the phosphate from this acid group onto ADP. That gives us an ATP and the product 3-phosphoglycerate 3-PG carried out by the enzyme phosphoglycerate kinase. That 3-PG molecule can then undergo what's referred to as a mutase reaction. And so the phosphate is moved from the 3 to the 2 position of glycerate. So 3-phosphoglycerate to 2-phosphoglycerate, carried out by phosphoglycerate mutase, often abbreviated PGAM. This 2-phosphoglycerate can then undergo a dehydration to- by an enzyme enolase to give the molecule phosphoenolpyruvate. . Phosphoenolpyruvate was the second of those useful intermediates that allow ATP synthesis despite the high ATP-ADP ratio in cells, and that reaction, as we already discussed, is carried out by pyruvate kinase and allows you to generate another ATP. Now, as I drew out the glycolytic pathway here, I also listed the delta G0 prime for every single step in the reaction. Remember, this is informative as to the equilibrium for each of these individual reactions. You'll note that for many of the steps, that delta G0 prime is positive. Remember what that means. That means that equilibrium favors the left. That is the reverse direction of the entire pathway. However, if you add all of these up, the delta G0 prime for the entire pathway-- so delta G0 prime for the entire pathway, glucose to pyruvate adds up to negative 6.9 kcals per mole. And so that negative 6.9 means that for the entire pathway, equilibrium favors the right. So glucose to pyruvate, the right direction. And that is why this can work as an overall pathway even though some of the individual steps, equilibrium may favor the opposite direction. Now you'll notice that there's several favorable steps where delta G0 prime is quite negative. Pyruvate kinase, phosphoglycerate kinase, hexokinase, phosphofructokinase. As we talked about before, these are able to pull the less favorable steps upstream forward by keeping concentrations of products such as 1,3-bisphosphoglycerate low, thus allowing this net reaction to move forward. You'll notice I drew three steps here as irreversible-- hexokinase, phosphofructokinase, and pyruvate kinase. Remember, these are all reactions in our-- there's no such thing as an irreversible reaction. There are some conditions where the reverse reaction, of course, can happen. Although remember, this-- when we discussed irreversible in metabolism, what we're referring to is irreversible under physiological conditions-- that is, conditions found in cells. You'll notice that those three steps are three of the four steps that have very negative delta G0 prime. And so you would expect that if we were going to reverse those reactions, you would need some kind of energy input, and you'll see later that these are the control points or also regulated steps in this pathway. And there are also the sites, just to give you a preview, that we're going to have to deal with if we're going to try to synthesize glucose, gluconeogenesis, the reverse of glycolysis.

~05:45Now of course, these delta G0 primes are very informative of what happens at equilibrium, but of course, what actually happens in a cell or in any condition, remember, is dependent on the equilibrium constant, but also on the conditions. Remember, it's delta G, delta G equals delta G0 prime plus RT times the log of the products over the reactants. And so delta G is what really matters for any individual reactions, and what's shown here on this slide is basically a approximate delta G0 change across the glycolytic pathway based on someone's approximation of the conditions found in cells. Now we can see and looking at this that there's really three big drops in delta G across the pathway. Here's the hexokinase reaction, here's the phosphofructokinase reaction, here's the pyruvate kinase reaction. And so it makes sense that these would be, then, the irreversible steps because there is what is actually pulling the pathway forward. It also makes sense that one would exert control at each of these steps, because this is what drives the flux of the pathway forward, but it's also where it's difficult to go back. Once you get here on this flat part of the curve energetically it doesn't matter so much which direction you go along the curve, but to try to climb back up each of these hills becomes difficult. And so we'll revisit this later in the lecture, but this is basically where the control points will end up happening in this pathway. Now, I want to note a couple other features about this pathway, and that is that we had to invest some energy early. You see you cost an ATP at the hexokinase and the phosphofructokinase step and then you harvest that ATP much later at the phosphoglycerate kinase and the pyruvate kinase steps. The textbook typically will split glycolysis into what they refer to as two stages, an investment stage and a harvesting stage, and that basically relates to that fact. And that's because you have a glucose molecule that then you use to generate two trioses. And then each of those trioses can be used to generate a pyruvate molecule. And so it costs 2 ATP to generate those to trioses, but then each triose generated allows you to recapture 2 ATPs. And so what that means, then, is that the net output from glycolysis is 2 ATPs. And so glucose going to two glyceraldehyde 3-phosphates costs you, whereas using those two glyceraldehyde 3-phosphates to generate two pyruvates allows you to recover 4 ATP for a net of plus 2 ATP molecules produced per glucose metabolized. Now if we add up the entire stoichiometry of what's going on here, we can also draw it another way, and that is we can basically have glucose plus 2 ATP molecules plus 2 inorganic phosphates, goes to 2 pyruvates plus 2 ATP, and that's because we're able to incorporate 2 phosphate molecules here at the GAPDH step. Now that's great, but if you're paying attention, you notice that we're missing one other thing in this stoichiometry, and that's that cofactor NAD that was also required to maintain electron balance across the GAPDH step. And so remember that for these pathways to work, we have to balance all things that are going in with all things are going out-- we can't create or destroy matter. And so those electrons have to be dealt with. When we discussed the GAPDH reaction last time, we needed that cofactor NAD in order to act as an acceptor for the electrons to allow that oxidative phosphorylation, if you will, of glyceraldehyde 3-phosphate to 1,3bisphosphoglycerate.

~09:22And so in order for this pathway to work, we need a way to regenerate that NADH back to an NAD in order to make electron balance. In other words, we have to deal with the fact that we've generated-- or that we required 2 NAD+ to run this pathway and generate 2 NADH. And so we need a way to get rid of those electrons from NADH to regenerate that NAD and allow the pathway to be balanced. Now it should be clear that this has to happen stoichiometrically for every single glucose molecule that goes through glycolysis. That is, we have to regenerate 2 NAD plusses for every glucose that we're going to convert into 2 pyruvates. And so for this NADH reoxidation to occur, we need a place to transfer those two electron pairs. And so just like any oxidation and reduction reaction, if we're going to oxidize NADH back to NAD+, something else has to be reduced, and that thing has to be reduced stoichiometrically with every single pyruvate that's produced from glucose and glycolysis. And effectively you will see that this is the role of fermentation. It's an ability for us to solve this redox problem, that we have to dispose of those electrons in the oxidation at the GAPDH step in order to deal with this electron waste of the pathway. Now, the useful way to do this is that we're generating a pyruvate stoichiometrically. And so we can use that stoichiometrically, that product pyruvate by reducing it to form the waste product, and that's effectively what fermentation is. It's turning that pyruvate into ethanol or lactate, which is reducing that pyruvate, picking up the electron waste from the NADH, and allowing you to regenerate the NAD that's required to run the GAPDH step. And so I'll just illustrate this. The simplest fermentation reaction that we can do is the run that's done by animals, and that's to generate lactate. And so to just illustrate this explicitly, here is-- so this here would be the reduced form of NAD, NADH. So this here this-- this is the nicotinamide group. Remember, it's hooked up to an ADP ribose to give nicotinamide adenine dinucleotide in the reduced form. So this can transfer electrons to pyruvate. Here's pyruvate. So as we showed last time, we can take 2 electrons from NADH reduced. That gives us this hydride ion, which can generate lactate. Plus, what we're left with is the oxidized form of the cofactor NAD+. And now this NAD+ can be reduced back to NADH at the GAPDH step, and the electrons from NADH, as it's being reoxidized to NAD, can go and take pyruvate and reduce it to lactate. So NAD gets reoxidized to NAD. Pyruvate now gets-- the ketone gets reduced to the alcohol as you go from pyruvate to lactate. Opposite of what's going on in the GAPDH step. There, the aldehyde is oxidized to the acid, while NAD+ oxidized goes-- is reduced to NADH. Here, NADH is reoxidized to NAD while taking the pyruvate and re-reducing it to lactate. This, by adding here, allows us now to regain balance across the entire pathway. This step is catalyzed by the enzyme lactate dehydrogenase, often abbreviated LDH. And that allows regenerating the NAD+ that's needed for the GAPDH step, which allows us to deal with the stoichiometry problem of the entire pathway. And so what fermentation does for cells is allows electron disposal by taking the product of the pathway pyruvate, reducing it stoichiometrically to form a reduced product-- as shown here, lactate, and that allows you to maintain electron balance across the entire pathway. And so this entire fermentation pathway, glucose to lactate, now allows the generation of the net generation of 2 ATP and run this pathway in cells so cells can maintain a high ATP-ADP ratio by fermenting glucose into lactate.

~13:14Now, if you're paying attention, this also now begins to describe why it is that oxygen is such a useful molecule for metabolism and actually why it's quite key to supporting the bioenergetics of cells. And so recall, oxygen was there when we burned wood and we used that example. And why oxygen is so important for burning wood, why it's important for doing glucose catabolism in our cells is that oxygen is a really great electron acceptor. That is, I can draw out oxygen can take 2 electrons that are generated as NADH waste, if you want to call it that, plus protons, and this generates water. And so an alternative to fermentation in order to allow glucose oxidation is rather than do fermentation, is to transfer those electrons to oxygen to run the pathway another way. In other words, if we write glucose to pyruvate and realize that this is an oxidation reaction, and so therefore, generates electron waste in the form of NADH that has to be recycled to NAD, we can do so by putting those reoxidizing NADH, means something else has to be reduced. This something else could be oxygen as a good electron acceptor to be reduced to water and find an alternative place to put those electrons. Or, if oxygen is not present, now we can do fermentation, turning pyruvate into lactate. It should be clear that if we want to completely oxidize glucose, as in burning wood-- that is, turn it-- all the carbon and glucose into the most oxidized form of carbon, CO2, this also requires places to put electrons. That is, we could generate more NADH. And of course, each of those electrons have to be dealt with. And the final home for those electrons needs to be something that's a good electron acceptor like oxygen, allowing you to continually reoxidize your NADH by reducing oxygen to water. So, in the absence of fermentation, oxygen can be used to maintain electron balance, and of course, much more energy is going to be released if we completely burn glucose than if we only partially oxidize glucose. And this is why oxygen allows more energy to be released, or, as you probably learned in high school, more ATP to be produced from glucose metabolism. Now the details of how all of this works are much more complicated than what I have drawn. Obviously fermentation happens the way I drew it, but all the other details about the role of oxygen and how it fits in to these pathways is more complicated than what I've drawn, and we'll cover this in the upcoming lectures. But the key concept is here, and that is the relationship between oxygen and fermentation is really what's illustrated here. These are two different ways that cells can use to dispose of the electron waste that's produced from carbon oxidation and glucose metabolism. Those electrons have to go somewhere. They can go to the product of glycolysis, say pyruvate to lactate, by lactate dehydrogenase, reduce the pyruvate to lactate, fermentation, or they can go somewhere else. A great electron acceptor like oxygen which allows these pathways to happen while saving the pyruvate to do something else, like be completely oxidized to CO2. Now it should be clear here, then, that one of the things that's special about fermentation is that absolutely no oxygen is required to do this, and this is part of the reason why this pathway is so ancient. Fermentation evolved in the pre-oxygen atmosphere, and that really is why it's such an ancient pathway. Only when oxygen levels rose in the atmosphere due to photosynthesis that enough oxygen was around that then other oxidative pathways could evolve as an alternative to fermentation.

~16:57Now, you'll note that the original description of fermentation by Pasteur was ethanol, not lactate. We'll cover the details of ethanol in a minute, but I just want to point out that making ethanol instead of lactate is simply an alternative product to dispose of pyruvate as a more reduced product. So ethanol is a more reduced product than pyruvate in that ethanol generation is an additional adaptation that allows you to dispose of the electron waste while also generating a molecule that's toxic, and therefore, allow organisms that make ethanol to better kill off their neighbors and their environment and compete for resources. But the overall concept is identical. Ethanol is really an alternative to lactate and fermentation but solves exactly the same problem. I want to talk about ethanol metabolism in more detail, but before we do that first I want to go through and just describe at a very high level some of the chemistry that allows all these reactions in glycolysis to work, because you'll see that the chemistry itself is not all that complicated even though we can draw it and it seems like somewhat overwhelming when drawn out as a whole pathway. Now I want to point out, we've already covered the chemistry for a lot of these steps. First of all, there's two dehydrogenase reactions. There's GAPDH and there's lactate dehydrogenase. Here's the mechanism for lactate dehydrogenase, it's a hydride transfer. The same thing happened at GAPDH with the additional details of adding the phosphate. We covered that previously in an earlier lecture. Note, we also covered before the chemistry of pyruvate kinase and phosphoglycerate kinase. In addition, there's two more kinase reactions that exist here, hexokinase and phosphofructokinase. These are really just straightforward phosphotransfer reactions using ATP to transfer a phosphate. Very straightforward, exactly what Professor Yaffe has already talked to you about, about how one does phosphotransfer for kinase reactions on proteins. Now, two other steps are isomerase reactions, triose phosphatase isomerase and glucose phosphate isomerase. We discussed this chemistry back when we did the sugar lectures. This is just acting on the open chain form of the carbohydrate and allows you to interconvert between the ketose and the aldose as I already drew during that lecture. And so that just leaves a few steps left to discuss that we can cover briefly. The first one that we'll talk about is this enolase reaction, which is simply a dehydration. I'll draw it here just to be explicit as to what's going on. So here is 2-phosphoglycerate. Simply dehydration to remove water, and that gives us phosphoenolpyruvate, PEP. The next step I want to discuss, phosphoglycerate mutase, is an example of a mutase reaction that is, remember, it's moving the phosphate from the 3 to the 2 position of glycerate. This is a class of reactions. It has somewhat of an interesting mechanism. And basically precedes by a phosphorylated enzyme intermediate. In the case of phosphoglycerate mutase, this is a histidine, and so there is a histidine with a nitrogen on it in the active site. And effectively, this nitrogen can pick up a phosphate and be primed via a reaction involving a molecule you've seen already from your discussion of hemoglobin with Professor Yaffe, and that's 2,3-bisphosphoglycerate. And so again, here's-- this is glycerate phosphorylated on the 2 and 3 position. So this is 2,3bisphosphoglycerate. And effectively, this molecule can bind in the active site and pick up a phosphate from either of the 2 or the 3 position. I drew it here is picking it up from the 2 position. That would, of course, generate a 3-phosphoglycerate. But more importantly, it ends up with the enzyme having this phosphorylated intermediate in the active site.

~20:39Once it has that phosphorylated intermediate in the active site, now it's ready to catalyze the mutase reaction. So I'll draw here first, this here would be 3-phosphoglycerate. Color the phosphate so you can see what's going on. This would basically, in the active site of the enzyme, pick up the phosphate from the active site and generate transiently this 2,3-bisphosphoglycerate intermediate, and then it can retransfer the phosphate from the other position back on to the active site of the enzyme so it's ready to carry out another catalytic cycle, and in the process, move the phosphate effectively from the 3 position to the 2 position. So you'll see that the phosphate is not actually being-- the same phosphate is not being moved within the same molecule, it's being ping-ponged back and forth off of this phosphate enzyme intermediate, and basically this is how cells catalyze various mutase reactions to move phosphates between hydroxyl groups. And so you have to prime this once with a separately-generated 2,3-bisphosphoglycerate, but once you have that phosphorylated enzyme in the active site, it can now continually ping-pong that phosphate around to effectively convert 3-phosphoglycerate to 2-phosphoglycerate, or an example of another mutase reaction, move the phosphate between two other hydroxyl groups on the same molecule. OK. Now the final reaction that we haven't talked about is this one, the aldolase reaction, which is a really key step in glycolysis because it splits that carbon-carbon bond to take the hexose-- fructose 1,6-bisphosphate, and split it into two trioses, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, which ultimately allows you to make this pathway work. And so I'll take you quickly through how aldolase works. So aldolase also has this amine in the active site. So here's FBP drawn in the open chain form. So you end up with this intermediate in the active site, which can undergo this chemistry to split this into the-- remove the lower half of the molecule. So you can see here, this generates this lower half of the molecule, glyceraldehyde 3-phosphate, as well as the top half, which can regenerate the active site, as well as the other product, dihydroxyacetone phosphate. And so very briefly, that's effectively the chemistry that allows aldolase to carry out this important reaction in glycolysis, splitting that fructose 1,6-bisphosphate into the aldose, glyceraldehyde 3-phosphate, and the ketose dihydroxyacetone phosphate. OK. So that is effectively the chemistry that allows you to turn glucose into pyruvate, as well as pyruvate into lactate, but what about making ethanol? How does that work? Now ethanol is an alternative fermentative product. And so the reactions to make ethanol is basically this. So here's pyruvate. First thing that happens is we lose this CO2. Without a change in oxidation state at the second carbon at the ketone in pyruvate. And so this ketone stays the same oxidation state, it's now an aldehyde. This is a molecule called acetaldehyde. And now we can pick up those two electrons from NADH and reduce this aldehyde to the alcohol, reoxidizing that NADH back to NAD+ and generating ethyl alcohol or ethanol. So, the fermentative part is turning the acetaldehyde into the ethanol because that allows you to dispose of those two electrons, reduce the aldehyde to an alcohol while reoxidizing the NADH to an NAD+, which is what allows glycolysis to continue so that NAD+ is available for the GAPDH step.

~24:01How this reaction works is-- I just drew it. It's exactly how the lactate dehydrogenase works. However, what we also now need to discuss is how you carry out this reaction, this carboxylation to take this CO2 off of pyruvate and make acetaldehyde. Now decarbonization reactions are really common reactions in metabolism. They end up being important for lots of pathways. Obviously if we're going to completely oxidize glucose into CO2, we have to remove CO2 for a molecule, and here's the first one-- first one that we're going to see for how that happens. Now there's two general mechanisms that metabolism uses for decarboxylation reactions, and I want to introduce the concept here of both of them. Now, the first one here is that these are referred to as decarboxylation reactions that occur in the context of either an alpha keto acid or a beta keto acid. Now, what do I mean by that? Well, pyruvate is an alpha keto acid, and that's because here's a acid group. The ketone is alpha to the carboxylic acid. This would be in contrast to this molecule. So I'll just draw with a generic R group. Where here, the ketone is beta to the carboxylic acid-- alpha, beta. So pyruvate is an alpha keto acid. This here is a generic beta keto acid. Now I want to point out that decarboxylations of bait ketone acids are very favorable, and they're favorable for the following reason. And that's because if I remove this CO2 from a beta keto acid, I'm left with this molecule, which hopefully you'll recognize as an enol. And just like we talked about for the pyruvate kinase reaction, enols much prefer to rearrange into the keto form. And so decarbonization of a beta keto acid generates an enol which will rearrange to the ketone. This becomes very favorable, and so it becomes favorable to decarboxylate a beta keto acid. Now, we can't use that exact same chemistry to decarboxylate an alpha keto acid. However, we can help that process, effectively mimicking the same thing that happens with beta keto acid, by instead introducing a cofactor. So I introduced the concept of cofactors when we talked about NAD and NADH. And remember, cofactors are molecules that provide functional groups that help facilitate the chemistry of various reactions. And so there's a cofactor that facilitates the chemistry that allows decarboxylation of alpha keto acids, and that cofactor is abbreviated TPP+, which stands for the thiamine pyrophosphate. TPP+, which is derived from the vitamin thiamine, sometimes also referred to as the vitamin B1. And so this is what TPP+ looks like. OK. So like many of our vitamins or cofactors, it's a complex molecule. So this is vitamin B1 or thiamine, and the cofactor TPP+ is basically the same molecule with a pyrophosphate group added on the end, TPP+. Now, to see what's going on, we're going to focus here just on this part, this reactive part of the molecule, which is right here, which for simplicity, I will just draw like this. So this here is just this basically part of the molecule here, that's the reactive part of TPP+. And what makes this cofactor useful is that this basically can exist in this stabilized carbanion form. This stabilized carbanion can react with alpha keto acid like pyruvate, shown here. And then this intermediate now becomes favorable for decarboxylation of the CO2 that's alpha to the ketone. And then here, we can regenerate the active TPP+ that we started with plus the other product, acetaldehyde. And then that acetaldehyde can then be reduced, regenerating NAD+ and giving alcohol for fermentation. OK.

~27:38Now incidentally, this is how you produce alcohol. How you metabolize alcohol is effectively reversing that last step and further oxidizing the acetaldehyde. And so this is ethanol. So the way ethanol is metabolized. And so the famous alcohol dehydrogenase enzyme basically does that reverse reaction, generates 2 electrons as a hydride ion. They get transferred to NAD+. That NAD+ gets reduced to NADH as this alcohol in ethanol is oxidized to the aldehyde in acetaldehyde. And this can then be further oxidized, that aldehyde to the acid. So here again, two electrons from the hydride iron can reduce NAD+ to NADH. And that generates, in the process, oxidizes the aldehyde to the acid. In this case, this is acetate. The acid form it would be acetic acid, also known as vinegar. And so effectively, the way you turn alcohol into vinegar is by the microorganisms oxidizing the alcohol they produce into acetic acid or vinegar. Now note, this is-- alcohol metabolism is two oxidation reactions. So we talked about last time, oxidation reactions are generally favorable, therefore energy is released. This is why alcohol has calories and it's impossible to make diet alcohol, because it will-- just no way around this. A couple other things about this, as I mentioned. This enzyme catalyzes. This is the famous alcohol dehydrogenase. This enzyme is an aldehyde dehydrogenase. The issue is is that this aldehyde hydrogenate is a less efficient enzyme than alcohol dehydrogenase, thus it can become rate-limiting for alcohol metabolism. And so if you overwhelm the system by drinking too much, effectively this enzyme can't keep up with this enzyme, you generate excess of the toxic acetaldehyde product. Acetaldehyde product is toxic, and this is effectively what generates hangovers.

~29:21It's also the case that there are individuals with polymorphisms of this aldehyde dehydrogenase. This was, I believe, discussed in Professor Yaffe's lectures on enzyme kinetics that have different catalytic efficiency at this step, and the people who have those polymorphisms can't metabolize alcohol as well. They tend to get flushing and whatnot because they build up this toxic acetaldehyde, and in fact, can be quite dangerous if they drink too much. Finally, this also illustrates effectively why when commercially you want to generate alcohol, like in beer or a wine, you need to keep oxygen levels low. Why is that? Because, well, the microbes will read their ethanol. It's a perfectly good source of calories for them as well because of these reactions. But they generate all this electron waste in the process. Those electrons have to go somewhere. Where do they go? Well, oxygen is a great electron acceptor. And so to metabolize this ethanol, they need to put those electrons somewhere. They need to put them on oxygen. And so oxygen is required to carry out this ethanol metabolism by the microorganisms. And so by keeping oxygen out of your fermentation of alcohol in commercial beer and wine production, it prevents the formation of aldehyde and acetic acid products which would be undesirable in your finished drink product. OK. Now this whole discussion also begins to suggest one big way in which glycolysis itself is regulated, and that's effectively by the availability of oxygen. And so to be explicit about this, I want to draw the relationship between fermentation, glycolysis, and oxygen in terms of maintaining electron balance across these pathways.

~30:58And so glucose is, of course, a major fuel for us. It's a major sugar in our blood. Yeast use a lot of glucose from the grapes in their environment. Plants, of course, make glucose as a storage carbohydrate. It's-- we discussed, starch is a polymer of glucose that uses glucose to survive the night. And effectively what they do is turn that glucose into two pyruvate molecules which can be used to generate net 2 ATP as we discussed. But creates this problem that it also generates NADH that needs to be recycled back to NAD+. In the absence of oxygen, you can ferment that pyruvate into lactate or alcohol or some other product that allows you to regenerate NAD+. Or-- and this is a pathway that requires no oxygen. However, that pyruvate can be further oxidized to CO2, but that's going to generate even more electron waste. That electron waste also has to be dealt with, and that requires oxygen or some other place to put those electrons, oxygen to water being a major driver of further oxidative metabolism. Now the beauty of fermentation as a pathway is that no oxygen is needed, but the trade-off is that it's much less efficient. You only get 2 ATP per mole of glucose that's-- 2 moles of ATP per of glucose that's fermented. You can imagine that a lot more energy is released-- if you can further oxidized glucose, you can generate, therefore, a lot more ATP if you use a complete oxidation of glucose, but this requires oxygen. And so a major way that glycolysis and certainly fermentation is regulated is that low oxygen is what promotes fermentation, whereas high oxygen suppresses fermentation, and that makes sense. It's intuitive. You get a lot more energy. If you completely oxidize your glucose, you can more efficiently get energy from oxidation of available reduced glucose carbon if you do it by oxidative pathways, and so therefore, in the presence of plenty of oxygen, you don't need to do fermentation. Whereas if you outstrip oxygen supply, as, say, might happen if your muscles are exercising, burning more ATP than can be kept up with by oxygen delivery from the blood, now you switch over to a more affirmative metabolism, something like lactic acid builds up that can then be dealt with later. Now, this fact that high oxygen can suppress fermentation is experimentally true in many organisms in many contexts. It's likely because the high ATP-ADP that's produced with oxidative glucose metabolism can directly affect some steps or deplete the NAD that's required to drive glycolysis. Exactly the mechanism that causes this is somewhat debated and likely depends on context and conditions. But it also sets out at least a way for us to begin discussing, how is it at a level of individual enzymes or reactions might something like high ATP-ADP ratio effect flux or flow through a pathway? All right. Well, let's just start by talking about what are the two things that could affect flux or flow through the pathway? Well, remember, thermodynamics, delta G, is what determines whether a pathway happens at all. And because delta G is proportional to things like the ATP-ADP ratio, you can imagine if this ratio gets too high, some of the steps might no longer be favorable to occur. That's certainly one possibility. But the other way that regulation can be done is actually kinetic, and this is because even though enzymes don't determine whether a reaction can happen, at least thermodynamically, they oftentimes do determine the rate of the reaction, and when we're talking about rate and flow through-- flux through pathways, flow through pathways, now rate can become very important.

~34:39And so the kinetics of enzymes, exactly what you talked-- heard about with Professor Yaffe, properties like V max or Km of an enzyme, which, of course, can be affected by allostery, and Professor Yaffe covered in great detail, these also, you can imagine, can affect how a pathway is regulated. And of course, things like ATP, ADP, and AMP as small molecules can be good allosteric affectors of some enzymes. And so let's now talk about, if we're going to regulate steps in a pathway, which steps should actually be regulated. And so shown here is, again, coming back to our delta G change map across glycolysis, as we alluded to earlier, the steps where it's hard to go back is this hexokinase step, this phosphofructokinase step, and this pyruvate kinase step, because these are where there's the big change in delta G that now makes it hard to reverse those steps. And it's exactly those steps that are regulated, because that makes sense. If this delta G is thermodynamically driving flux through the pathway, changing the rate of the enzymes that catalyze those steps is also going to change the rate at which the pathway as a whole can happen. Now as a result, it's these steps, hexokinase, phosphofructokinase, pyruvate kinase, that are often referred to as the rate-limiting steps of glycolysis. Personally, I don't like referring to memorizing things like these are the rate-limiting steps of the pathway, because in reality, what step of any pathway is going to depend on the context. However, often this concept of rate limitation when people refer to pathways really is coming from this energetic argument about which steps have the biggest drop in delta G, and it's important for you to understand that, not just memorize what steps are rate-limiting. Now I also want to point out that these steps actually are in interesting places in glycolysis. So this is basically entry into the pathway, and this is exit from the pathway. And this makes sense, too. You don't want to start committing carbon into a pathway unless you're actually going to need it, and you also need to match output to input. And what you'll see is that many steps in pathways that are regulated, in addition to being the ones with the biggest delta G changes, are also often the entrance and exit from the pathway, which, of course, is the case in glycolysis, but also in most pathways it makes a lot of sense just from logical considerations. All right. Now if we're going to control things that the level of enzymes and rate, how can we control rate of a reaction? That is, how can we control enzyme kinetics? Well, if you remember from Professor Yaffe's lectures, you can increase the rate of a reaction-- that is, catalyzed by an enzyme-- by increasing the Vmax of that enzyme; or, if the substrate concentration is close to Km, by lowering the Km of that enzyme. How can you decrease the rate? Of course, you can do the opposite. You can decrease the Vmax; or, if the substrate concentration is close to Km, you can increase the Km. So how can I change these things? How can I alter Vmax or Km of an individual enzyme? Well, there's a number of ways I can do this. One is is I can make more enzyme. What will making more enzyme do? Well, effectively that will increase the Vmax of an enzyme. Or if I get rid of enzyme, that will decrease the Vmax of an enzyme. I can use a different version of the same enzyme. That is, have an enzyme like pyruvate kinase but have it come in a bunch of different varieties, have different genes encode different pyruvate kinase isoforms such that those isoforms have different Vmax and Km relationships with respect to substrate, and effectively, by using a different version of the enzyme, a so-called isoform of an enzyme, you can now have different properties.

~38:38Now you'll note that the change enzyme kinetics this way basically is new transcription translation. That's fairly long on the timescale of needing to adapt to metabolism, but that can work. But sometimes a lion's attacking you, you need to run away quickly, you may not have time to make more enzyme. You also need direct control of the enzymes, and this is where allosteric regulation of enzyme function comes into play. Allosteric regulation can be a result of signaling. Phosphorylate, acetylate an enzyme, that can change its properties. Or you can have metabolite binding. Bind ATP, bind AMP into some allosteric site that can change Vmax or Km and operate on a short time scale to allow metabolism to adapt in a more acute setting. I want to say that time scales matter for real biology if you want to understand physiology and how it applies to how metabolism and physiology relate to biology that you'll encounter in other contexts. You realize that some responses need to be acute. Those allow you to adapt to conditions quickly, be able to get enough ATP to run away from the lion, et cetera. But you also need adaptations that work on longer time scales, because it's things like this that allow you to enact programs that allow you to adapt long-term to whatever new conditions a cell may face in its environment. All right. Now, before I get into talking exactly how these things actually work to regulate glycolysis, I want to discuss a few details about regulation of pathways in general. And while we talked about these with respect to glycolysis, these points will end up coming up over and over again and really apply to other pathways. Now I may not explicitly point this out, but the same considerations for how glycolysis works, how it's regulated has to be true for all metabolic pathways. And remember, any pathway is just variations on relatively few chemical-- chemistry reactions that are repurposed in a way to build some other thing that's useful to the cell, and you'll see this over and over again. Now, it has to be true for every pathway that it has to be thermodynamically favorable, and that means either that the pathway itself, glucose to pyruvate, is thermodynamically favorable, or it's coupled to some kind of energy input, like ATP hydrolysis that allows the pathway to be favorable. Every pathway is going to be built in a way that intermediates are generated along the way that allow that pathway to accomplish its goals. In the case of glycolysis it was the two steps that allowed us to synthesize and incorporate phosphate despite the high ATP-ADP ratio in cells. And in all cases this has to obey the laws of thermodynamics. That is, we have to obey constant conservation of mass. That means we have to account for all carbon, all electrons, delta G has to be less than 0 across the entire pathway. You'll see that the regulated steps will often be those with the largest thermodynamic considerations, like the big drops across hexokinase, phosphofructokinase, and pyruvate kinase. They'll often be at the entrance and exits to pathway because you want to regulate, you don't want to commit something to a pathway if you're not going to use it, and you want to match input and output. And all of this is going to have regulation that acts on both short and long time scales so it allows it to match physiology. All right. Now with that in mind, now let's discuss how we can regulate glycolysis using both short and long time scales as-- using really short time scales, allosteric regulation of glycolysis in a way that makes it most useful to cells. All right. So the first step is uptake of glucose into the cell, which in animals, like us, is passive, but can still be regulated by glucose transporters, often regulated glute proteins. And it's really these glucose transporters that can often determine whether glucose is actually taken up into cells.

~42:39A famous example of this is FDG PET scanning, which is used to measure glucose uptake into tissues in humans, where basically a hydroxyl group is replaced with a positron-emitting fluorine atom, fluorodeoxyglucose, that is taken up and phosphorylated in cells, and it's this lighting up of this on a positron-emitting scan you can tell that which tissues in the body take up glucose and that's a property of cancer, and so it is often used as a way to detect or stage cancer. Another example of this is insulin. Basically one of the major actions of insulin on your tissues is that when glucose is high, your body makes more insulin. That causes glucose transporters to be put on the surface of your muscle and your fat, which allows that glucose now to get into those cells and effectively allows those cells to dispose of the glucose in the blood and keep glucose in the right physiological range. And so uptake of glucose, at least in animals, is one important step that is really controlled largely by whether or not there is a transporter on the surface. All right. Once that's trapped as-- sorry, once it's in the cell as glucose, hexokinase then traps that molecule as glucose 6-phosphate. And it turns out the glucose 6-phosphate is an inhibitor of hexokinase. Why is that? Well, we will see in the next lecture that glucose 6-phosphate can be turned into glycogen in the case of mammals or starch in the case of plants. And so glucose 6-phosphate, if you're going to store glucose, you can ship it off into glycogen, or you could commit it further down into glycolysis. But if you don't need it for a glycolysis and your stores are full, you'll build up glucose 6-phosphate, you want to inhibit hexokinase to keep things from flowing through the system. The next step that's regulated is phosphofructokinase. So phosphofructokinase, of course, makes FTP. The step downstream of that that's regulated is pyruvate kinase, PEP to pyruvate, and it turns out that FTP will stimulate pyruvate kinase, whereas PEP will inhibit phosphofructokinase. Makes sense. You want to match input to output. If you have a lot of FTP, activate the enzyme to dispose of it. If you're building up PEP, tell you to stop sending molecules down the pipeline. It turns out, PFK is actually the major regulatory step of glycolysis, because this is really what commits that carbon to going through glycolysis as opposed to upstream of that, it could be stored or not. So it's really that step that's a commitment step. And so it's also regulated by some of the outputs of the pathway. And so some of those outputs are ATP downstream of pyruvate, which we'll talk about later in the course. There's some other molecules. Alanine, citrate. It turns out that high levels of citrate will also inhibit PFK. High levels of ATP will inhibit phosphofructokinase. Whereas high levels of AMP will activate phosphofructokinase. Makes sense. A major output of glycolysis is to keep the ATP-ADP ratio high, the energy charge high in the cell. If the energy charge is very high, high ATP, shut off phosphofructokinase, don't need to do more glycolysis. If the energy charge low, high AMP, activate phosphofructokinase. If production of other molecules downstream like citrate are high, come back and stop sending things into the pathway. Turns out, alanine can inhibit pyruvate kinase, another step downstream. Great. This is effectively general regulation, allosteric regulation of how glycolysis works. And what I wrote is was generally true across different tissues, but of course, these regulations can be tweaked to be more or less important to match physiology and function. Now I know many of you are going to take the MCAT. And while I would never want to teach just to take a test, a couple of things that you should remember for that test is phosphofructokinase is a major regulator. Its big allosteric regulators are AMP, as well as ATP and citrate.

~46:38Whereas the other steps, hexokinase, you should know that it's regulated by-- negatively regulated by glucose 6phosphate, and pyruvate kinase is positively regulated by FBP. Now one other point that I want to mention, just because it will come up sometimes on MCAT exams, is that there's an additional detail about how PFK is allosterically regulated, and that's because there's this side reaction where fructose 6-phosphate can be phosphorylated on the 2 rather than the 1 position to make this molecule, fructose 2,6-bisphosphate. This is a reaction that is catalyzed by bifunctional enzyme called PFK-2/FBPase. So basically phosphofructokinase on the 2 position, FBPase, we're moving it from the 2 position which allows you to regulate the levels of fructose 2,6-bisphosphate. It turns out that PFK in glycolysis, which, of course, generates FBP, is positively regulated by fructose 2,6-bisphosphate. And the production of fructose 2,6-bisphosphate is something that can be inhibited by ATP, inhibited by phosphoenolpyruvate, and activated by ATP. So exactly what is shown here is allosteric regulation. Just some of this allosteric regulation is via this side enzyme to make a separate product, fructose 2,6-bisphosphate, and there's control theory considerations for why that might happen. All right. So at this point, we've learned how we can start with glucose, oxidase that glucose via a pathway that now allows us to get the energy that cells need to maintain ATP-ADP ratio in a good physiological range despite the high ATP-ADP ratio in cells. However, it should also be clear that in order for this to work, we need a way to actually get that glucose from somewhere to begin with. You probably learned in high school that plants use photosynthesis as a way to get energy from the sun. Well, the synthesis part of photosynthesis is generating the sugar that those plants can then use to burn and keep ATP-ADP high at night and survive the night until the sun comes up again when they can again use energy from the sun directly to get energy. We as animals evolve the way that we have to eat the plants. We have to get the glucose that the plants make, but of course, we also use-- generate-- have the ability to generate some glucose, and this is best illustrated from muscle physiology. And that is, we alluded to earlier, that if we're running away from the lion, our muscles might outstrip the supply of oxygen in the blood and basically generate a whole lot of lactate. Well, that lactate is perfectly good fuel. Just like the yeast, can oxidize or we can reoxidize ethanol. We can also use lactate as a fuel, but it turns out, a big job of our liver is to take that lactate from the blood, as well as other molecules, and regenerate glucose so that our blood glucose remains in a constant level. This cycling of glucose and lactate across the body between the muscle and the liver is sometimes referred to as the Cori cycle and involves a clear set of reactions to turn that lactate back into glucose, a process called gluconeogenesis. And unfortunately we're out of time today, but next time I will start off the lecture by going through how it is that gluconeogenesis works to turn something like lactate back into glucose so that organisms have the glucose to begin with to run glycolysis. Thank you.

2.2获能期核心机制（第 6–7 步）：GAPDH 硫酯能量捕获、第一次底物水平磷酸化与三大经典抑制剂

一、碳的氧化态阶梯与辅酶 NAD⁺ 的氢负离子（H^-）搬运术

进入糖酵解第 6 步之前，Vander Heiden 教授停下脚步，在黑板上画出了全书最核心的一条生物能学公理：生物体内的分解供能（Catabolism），化学本质就是把碳原子上的电子一步步转移给电负性更强的氧原子（碳的逐步氧化）；碳原子的还原度越高，氧化时释放的自由能就越多！让我们看看碳原子的五级氧化态阶梯：

-CH_{2-CH2-(饱和烷烃（脂肪烃链/汽油，最还原，能量密度最高）)} ──(-2e^-)──► R-CH(OH)-R'_{(醇基（糖类主碳链）)} ──(-2e^- (H^-))──► R-CHO_{(醛基/酮基（羰基）)} ──(-2e^- (H^-))──► R-COOH_{(羧基（有机酸）)} ──(-2e^-)──► CO_{2(二氧化碳（燃尽态）)}

这就是为什么在日常生活中，燃烧汽油（全饱和碳氢链）比燃烧木头（纤维素，多羟基碳水化合物）每克释放的热量高得多；在营养学上，1 克脂肪彻底氧化释放约 38 kJ（9 kcal）能量，而 1 克糖或蛋白质只能释放约 17 kJ（4 kcal）能量——因为脂肪的碳处于最还原的 -CH_2- 状态，而糖分子里除了一个羰基外，其余碳都已经带有一个羟基（被部分氧化过了）！

现在回到糖酵解：我们要在获能期合成 ATP，就必须先造出像磷酸肌酸那样能把磷酸基团塞给 ADP 的高能磷酸化合物（即 1,3-二磷酸甘油酸 1,3-BPG 和 磷酸烯醇式丙酮酸 PEP）。可是葡萄糖本身没有磷酸，前 5 步装上去的两个磷酸又全是从 ATP 借来的（如果只靠它们还债，净收益只能是 0）。要想净赚 ATP，细胞必须想办法把细胞质里游离的“免费无机磷酸（P_i）”装到三碳糖上，而且不许消耗 ATP！这笔装磷酸的能量从哪来？只能来自把 3-磷酸甘油醛（G3P）的 C1 醛基氧化成羧基所释放的氧化还原自由能！

但在生物学里，物质与电荷必须严格守恒。当 G3P 的醛基（-CHO）氧化为羧基时，脱下的两个电子是以一个 氢负离子（Hydride ion, H^- = H: = 1H⁺ + 2e^-） 的形式离开碳原子的。水溶液里绝不可能存在游离的 H^-，这两个电子必须立刻交给一个专门的“电子搬运工”——这就是由 维生素 PP（又名烟酸/尼克酸/烟酰胺，即维生素 B₃，缺乏时引起糙皮病 Pellagra） 衍生的核心脱氢酶辅酶：烟酰胺腺嘌呤二核苷酸（Nicotinamide adenine dinucleotide，简称 NAD⁺ 或辅酶Ⅰ） 2015·名解！

3D3D 分子：烟酰胺腺嘌呤二核苷酸（NAD⁺ ⇄ NADH）步骤 2–3：吡啶环 C4 位接受氢负离子 H⁻，还原态在 340 nm 具特征紫外吸收峰↗

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二、第 6 步与第 7 步：GAPDH 的有机化学魔术与第一次底物水平磷酸化

理解了碳氧化放能与 NAD⁺ 接电子之后，我们来看全章最精彩、MIT 7.05 必考的化学机制——第 6 步：3-磷酸甘油醛脱氢酶（Glyceraldehyde 3-phosphate dehydrogenase, GAPDH） 催化的氧化磷酸化偶联反应 2024·单选：

3-磷酸甘油醛（G3P） + NAD⁺ + P_i ⇌(3-磷酸甘油醛脱氢酶（GAPDH）) 1,3-二磷酸甘油酸（1,3-BPG） + NADH + H⁺ (Δ G'^° = +6.3 kJ/mol)

请思考：如果让醛基直接被 NAD⁺ 氧化成游离的 3-磷酸甘油酸（3-PG），虽然极度放能（Δ G'^° = -43.0 kJ/mol），但所有的自由能都会以热量的形式白白散失，随后再想把无机磷酸 P_i 加到羧基上生成 1,3-BPG（Δ G'^° = +49.3 kJ/mol）就难如登天了！那么，GAPDH 是如何像一位绝顶聪明的化学家一样，把 -43.0 kJ/mol 的醛基氧化能量一滴不漏地锁进分子内部，用来驱动 +49.3 kJ/mol 的无机磷酸加成呢？请看下图的四步催化机制：

紧接着，刚出炉的 1,3-BPG（C1 位是一个由羧酸与磷酸缩合而成的混合酸酐——高能酰基磷酸键，水解 Δ G'^° = -49.3 kJ/mol 2008·单选）立刻进入 第 7 步，在 磷酸甘油酸激酶（Phosphoglycerate kinase, PGK） 催化下将 C1 高能磷酸基团直接转移给 ADP：

1,3-二磷酸甘油酸（1,3-BPG） + ADP ⇌(磷酸甘油酸激酶（PGK）, Mg²⁺) 3-磷酸甘油酸（3-PG） + ATP (Δ G'^° = -18.5 kJ/mol 或 -4.5 kcal/mol)

这里藏着四个华农考研必考的黄金结论，请你逐条标亮记牢：

• 名解必背：第 7 步（PGK）是糖酵解中的 第一次底物水平磷酸化！底物水平磷酸化（substrate-level phosphorylation）：指底物分子因脱氢氧化或脱水导致分子内部能量重新分布，形成高能磷酸键（或高能硫酯键），随后在激酶催化下直接将高能磷酸基团转移给 ADP（或 GDP）生成 ATP（或 GTP）的过程；该过程与呼吸链电子传递无关，不需要氧气参与 2023·名解。
• 热力学偶联拉动：虽然第 6 步（GAPDH）本身的 Δ G'^° = +6.3 kJ/mol（略微吸能），但第 7 步（PGK）强力放能（Δ G'^° = -18.5 kJ/mol），把 1,3-BPG 一生成就立刻抽走变成 3-PG，两步偶联合计 Δ G'^° = +6.3 + (-18.5) = -12.2 kJ/mol，完美驱动反应滚滚向前！而且注意：第 6 步（GAPDH）和第 7 步（PGK）在生理条件下均是可逆反应，糖酵解与糖异生完全共用这两步酶（PGK 之所以按逆反应命名为“磷酸甘油酸激酶”，正是因为在糖异生中它消耗 ATP 磷酸化 3-PG） 2013/2014/2016·单选！
• 能量收支平衡点：因为 1 分子葡萄糖生成 2 分子 G3P，所以在第 7 步生成了 2 × 1 = 2 ATP，恰好将准备期（HK 和 PFK-1）借贷消耗的 2 分子 ATP 全部还清，此时净产 ATP 刚好归零（= 0）！
• 红细胞内的 2,3-BPG 支路（Luebering-Rapoport 支路）：在没有线粒体的成熟哺乳动物红细胞中，有一部分 1,3-BPG 并没有走 PGK 生成 ATP，而是被 二磷酸甘油酸变位酶（BPG mutase） 催化将 C1 的磷酸移到 C2，生成 2,3-二磷酸甘油酸（2,3-BPG，浓度高达 4～ 5 mmol/L），随后再由 2,3-BPG 磷酸酶水解回 3-PG 重返糖酵解。2,3-BPG 支路虽然跳过了 PGK 这一步底物水平磷酸化（少赚了 ATP，但也避免了红细胞内 ATP 过高反过来抑制 PFK-1），其核心生理意义是生成 2,3-BPG 作为血红蛋白（Hb）的别构调节剂：结合在脱氧 Hb 四个亚基中央的正电荷空腔内，稳定 T 态（紧张态），降低 Hb 对 O₂ 的亲和力（使氧解离曲线右移），帮助红细胞在组织毛细血管中高效卸氧 2017·简答！

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三、经典生化实验剖析：砷酸盐解偶联、碘乙酸与氟化物三大抑制剂

在国内外生化考试中，围绕第 6 步（GAPDH）和第 9 步（烯醇化酶）设计的三大经典毒物/抑制剂实验，是考察你是否真正听懂化学机制的试金石：

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🎧 MIT 7.05 课堂原声英文实录对照（本节关联 1 讲 · 完整逐段实录）点击展开 / 收起 ▾

🎬 MIT 7.05 Lec 20 · Bioenergetics & Glycolysis I (Prof. Vander Heiden)⏱ ~50:00

~00:00MITOCW | 20. Bioenergetics/Intro Pathways/Glycolysis I PROFESSOR:

~00:02Today we're going to discuss bioenergetics, continue that discussion and use that as a transition into also beginning to see how that applies to understanding glycolysis, the pathway that allows glucose breakdown. So just as a reminder of a couple of the points that we discussed last time, so last time we discussed what makes reactions favorable. And so remember, you could have any reaction, A to B-- A to B could be a single step. A to B could be an entire pathway, all steps in the pathway, that whether or not that reaction or pathway happens is determined by thermodynamics. It's not determined by enzymes. Enzymes are important, of course, to control the rate of reactions so they may allow things to happen that otherwise wouldn't happen. But whether the reaction happens or not is determined by free energy. And so free energy, delta G, remember we discussed is the following relationship. So it's related to this constant delta G0 prime, which is related to the equilibrium constant for any pair of products and reactants as well as this formula rt times the log of the products over the reactants. And so remember this means that whether a reaction happens depends on the equilibrium constant, but it also depends on the actual conditions present. That is how much substrate and how much product is there. And so what this means is that biology can come up with ways by keeping the product concentrations low to do things that may move in the opposite direction of what you would predict from equilibrium. And also by coupling these less favorable reactions to more favorable reactions, you can also allow biology to do unfavorable stuff. And this is, of course, where the role of ATP comes in. And we discussed this at the end of last time as well. Of course, what's important then becomes the ratio of ATP to ADP. And to illustrate that, we use this example. We use the-- remember ATP hydrolysis has a delta G0 prime of minus 7.5 kcals per mole. That, of course, tells you this is about the equilibrium constant. That tells us the equilibrium lies to the right. We can couple it to this first reaction in glycolysis, which traps glucose in the cell by adding a phosphate group. Delta G0 prime for this is positive 3.3 kcals per mole. This positive number tells you that the equilibrium lies towards the left. But when we couple these two reactions together, we add these two numbers together, and now we get a delta G0 prime of minus 4.2 kcals per mole, which are the coupled reaction. The equilibrium lies now again to the right. And that's how we can use ATP hydrolysis in order to carry out this unfavorable process, trapping the glucose in the cell.

~02:38Now how much glucose will be trapped? Well, of course, that will come from this reaction up here. And that's because whether or not a reaction occurs or not of course depends not on the equilibrium constant but on the actual conditions and the delta G of that reaction.

~02:54And so we can just plug that in for this system. And so we see delta G0 prime minus 4.2 kcals per mole. That says equilibrium lies to the right. But how far really depends on the conditions. And that's defined by this ratio of products over reactants. And you can imagine that this term here, as long as it's less than 4.2, you will favor glucose trapping inside the cell, whereas, of course, if it's greater than 4.2, then you'd favor-- the opposite direction would be favorable. Now this is really critical because this really explains how it is that ATP actually works to help cells do various reactions. And so if an ATP-ADP ratio exists such that it can make delta G less than 0 for the reaction that it's coupled to, that is how it provides the energy that then allows unfavorable reactions to occur. And so please do not make the common mistake that many, many biologists make when they state, oh, ATP equals energy. There's a lot of ATP. The cell must have a lot of energy. Remember it's the ratio of ATP to ADP that provides the energy. And so that means ATP can be sky high, but if ADP is equally high, there's no energy there. Consequently, ATP can be very low. But if ADP is extremely low, now you still have a high ATP to ADP ratio. And that is what provides the energy. And that's because this occurs regardless of concentration. It's because it's this ratio of ATP to ADP that ultimately says how much energy is there for the ATP to drive a reaction. In other words, delta G, or whatever the reaction is coupled to, is going to be proportional to the ratio of ADP to ATP. Great. Now how much ATP do you need then? What ratio do you need to drive a reaction? Well, it's hard to say that in absolute terms because it's going to be defined by how that ratio is coupled to the ratio of whatever other reactants and products. It also tells you, remember, as we talked about before, we can switch the directions of these reactions and we just flip the signs. The directions here are arbitrary. And so that means it also tells us how much energy we need to put in if we're going to charge up an ATP-ADP ratio. In other words, if we want to synthesize ATP, that is take ADP plus phosphate and make an ATP, well, what's this? What we just-- the exact opposite sign of that delta G0 prime is going to now equal plus 7.5 kcals per mole. And so that means that the equilibrium of this reaction is going to lie to the left just as that one lied to the right. And it's going to lie to the left by that much. And if we want to know how much energy it then takes to make ATP, well, obviously, it's not going to happen because equilibrium favors this direction. It's not spontaneous in that direction. But at various conditions, we can know by plugging in our formula delta G equals delta G0 prime plus 7.5 kcals per mole plus RT times the log of ATP. So if I have my ADP-- I'm sorry-- my ATP concentration sufficiently low relative to my ADP concentration, I can still make this reaction happen. Now that's not going to give you an ATP-ADP ratio that is actually consistent with being able to do anything else into the cell. And so but what it says is that if I am able to couple energy from nutrient metabolism to this reaction and overcome this positive 7.5 kcals per mole, that basically will allow me to now synthesize ATP and maintain an ATP-ADP ratio in the cell that allows cells to carry out other functions by the same rules that we talked about that would be unfavorable. Now there's lots of implications here for metabolism, because it says how much input you need to synthesize ATP. How much energy you need to synthesize ATP is going to depend on how charged this ratio is. And how useful that ratio is to catalyze other reactions is also going to depend on how charged that ratio is.

~06:50And in essence, this is why we cannot store energy as ATP. This is why we didn't eat ATP for breakfast and instead ate cereal that has a bunch of glucose in it, because we can use that glucose to get ATP but the ATP itself is not something that we really can burn to get energy in a sustained way. In other words, the delta G of ATP synthesis, making ATP is going to be proportional to the log of the ratio of ATP over ADP for all the reasons I described. The delta G of using ATP, ATP hydrolysis, using ATP to drive reactions is also proportional to RT times the log, in this case, of the ADP to ATP ratio. And so because you need to consume this ratio to get energy, and you need to put in energy to create this ratio, you are constant-- and how much is an exponential property-- you are constantly having to generate ATP in a way that allows you to then have a ratio that can then be consumed to fuel other reactions. And that's what's shown here on the slide. So I just graphed here what the ATP-ADP ratio is relative to a theoretical delta G that's shown here as a linear scale, here on a log scale, that basically there's this logarithmic relationship and so you need an exponential increase in energy input to drive this ratio up higher in order to, quote unquote, "store" more energy that can be used for later. And so this system is not one that you can use as an energy storage system and ultimately explains why cells have to do constant metabolism, have to constantly do some sort of metabolism to keep this ATP-ADP ratio charged for it to be in a useful range to have an ATP-ADP ratio that can be coupled to drive otherwise unfavorable reactions in our cells. So to say this another way, our cells need to constantly catabolize nutrients, burn glucose, burn some other fuel. They have to do this because they have to keep that ratio charged in a range where that can then support other cell functions, allow them to fight entropy and maintain order in biology. We alluded last time if you stop making ATP, well, what's going to happen? Well, that ATP-ADP ratio the cell has is going to fuel whatever reactions it can do until suddenly that ratio falls below the point where it's useful to drive those unfavorable reactions. In fact, if you stop ATP production, you will consume that ATP-ADP ratio in seconds. And this is why if you poison ATP production, cells die very quickly. Remember last time we mentioned you have a heart attack, blood flow stops going to a tissue. No more nutrients and oxygen. Cells die quickly. Cyanide poisoning does the same thing. Cells die quickly if you stop their ability to maintain this ATP-ADP ratio in the right range. And so remember the concentration, the absolute concentration of ATP for these reasons is not a good indicator of cell energy. The correct indicator of cell energy is the ratio of ATP to ADP. This term is sometimes referred to as the energy charge. And so that can be the ATP to ADP ratio, or oftentimes more correctly is the ATP to AMP ratio. Why the ATP to AMP ratio? Well, that's because once you understand this, you can now understand that cells have a failsafe mechanism to protect themselves if this ratio of ATP to ADP becomes too low. What is that mechanism? Well, you can carry out this reaction. Take two ATP molecules and turn them into transfer a phosphate to make an ATP plus an AMP. Why is that a failsafe mechanism? Well, if they care about this, if the ATP-ADP ratio is what maintains your ability to drive these thermodynamically unfavorable reactions, if I sacrifice the denominator by removing an ATP and get an ATP out of it, I've now at least temporarily fixed that ratio. Now this is something that obviously cannot go on forever.

~10:36But in the short term, it does allow cells to protect that ratio. And in fact, what cells actually defend is this ATPAMP ratio. And it's very, very difficult to change the ATP-AMP ratio in a cell and actually have it live. So it's really this ATP-AMP ratio that is most formally related to what would be called energy charge. But this is really why this is important is for all the reasons that I just described. All right. So hopefully, it's clear to you now why cells have to keep ATP-ADP ratio in the right range, why this is useful for them to now carry out all of these unfavorable reactions-- fight entropy, stave off death. And they can even have a failsafe here and sacrifice ADP as a way to try to maintain that as a very last resort. It also says why cells have to always do catabolism, because you always need the energy input to keep that ratio in the right range. However, you might imagine that, in real life, the messiness of biology, that there's lots of situations where the demand for ATP will suddenly increase. Well, of course, maybe you can just metabolize more sugar, do more nutrient catabolism to try to keep that ratio high. But that takes time. And so you need ways for cells to quickly respond to then allow them to mobilize all of this extra thing to-- extra fuel to do that. For example, we're walking around our ancestors on the plains somewhere and a lion appears. We don't have time to mobilize a bunch of new fuel. We have to be able to run away really quickly or the lion's going to eat us. And so cells have systems to suddenly respond to I need a lot more ATP demand to run away by having ways that one can buffer this ATP-ADP ratio, protect it in the short term, and allow cells time to ramp up nutrient metabolism in order to keep this high. All right. Well, if we wanted to create such a buffer system, how would we actually do it? Well, obviously, we have to follow all the rules of thermodynamics that we discussed. And we have to do so in a way that actually illustrates how it is that very well how metabolism works. In other words, if suddenly the ATP-ADP ratio drops, if we have a sudden drop in ATP-ADP ratio because we have increased demand, needing to run away from a lion, how could we actually create a buffer system that protects this? Well, to do it, we would need to couple ATP-ADP to some other product reactant pair, let's call them x and y, such that the ratio of y to x is coupled to the ratio of ATP to ADP. And you want it coupled in such a way that if that ratio goes down, this ratio can now protect ATP, basically favor ATP to ADP synthesis. However, when nutrients are plentiful and this ratio is really high, it then in turn can charge up this ratio so that it can buffer it later when the ATP-ADP ratio starts to fall. Now the most famous example of this actually comes from muscle physiology. And it's best described in terms of muscles and how they quickly respond to increased demand, like that running away from a lion. However, it in fact applies to many, many cells, including most non-muscle cells in animals.

~13:47All right. And I should say that it turns out that if you turn off ATP synthesis in a muscle, people have estimated that your ATP-ADP ratio will fall into a range where it is no longer useful to do anything within a matter of seconds, some claim even less than a second. And so this system ends up being very important.

~14:08Now the system that muscle uses, and many other cells do as well, is something called the creatine/creatine phosphate system. Now if you guys are weightlifters or bodybuilders out there, you probably have heard of creatinine because it's a very popular supplement that is taken for weightlifting, something I obviously don't do a lot of. But that creatine/creatine phosphate is out there. I'll describe to you what it actually does for muscle cells. And you can decide for yourself if this is actually useful as a energy or weightlifting supplement. So what is creatine/creatine phosphate? What does it look like? Well, it's basically looks like this. By the way, I will use this abbreviation often throughout this course, just a shorthand to draw a lot of these structures. So this here would be a carboxylic acid group drawn like this or like that, drawn to the carboxylic acid group just so you understand my shorthand. OK. So this here is the molecule creatine. If I put a phosphate here, this now becomes creatine phosphate. Again, just to remind you, this P with a circle over it is a phosphate group. I'll draw this particular phosphate group because it's a nitrogen-phosphate bond, a phosphoramidate bond. This is actually a bond that is more labile than the PO bonds that we're used to seeing in the phosphodiesters like ATP, et cetera. All right. So that's creatine and creatine phosphate. And if we draw out the reaction, creatine phosphate goes to creatine plus inorganic phosphate, so hydrolysis of creatine phosphate. This has a delta G0 prime of minus 10 kcals per mole. What does that mean? It's negative. Tells us about the equilibrium constant. Equilibrium lies to the right. Remember ATP to ADP hydrolysis shown over there, that hydrolysis has a delta G0 prime of minus 7.5 kcals per mole. Equilibrium also lies to the right. This number is more negative. This equilibrium lies further to the right than ATP hydrolysis does. So now what happens if we couple this reaction to the ATP hydrolysis reaction? So now we can do ATP plus creatine goes to creatine phosphate plus ADP. So we've coupled those two reactions together. So delta G0 prime, ATP to ADP is minus 7.5. Creatine phosphate to creatine in that direction is negative 10. In this direction, it would be positive 10. And so as I've drawn the reaction, this delta G0 prime will be plus 2.5 kcals per mole. 10 minus 7.5 plus 2.5, which tells us then that this equilibrium now lies to the left. However, which direction this reaction actually goes is not determined only by the equilibrium. It's really determined by delta G. So we can calculate that. So delta G equals delta G0 prime plus RT log of, as we've drawn it, creatine phosphate over creatine and ADP over ATP. So you can now imagine that this term here is the sum of those two. So it's plus 2.5. And so if this term is less than 2.5, what does that mean? Well, if it's-- I'm sorry. Less than negative 2.5, then delta G will be negative and you will favor creatine phosphate production. If this term is greater than negative 2.5, then delta G will be positive and you will favor ATP production. Why is that important? Well, if you think about the conditions, how you could make this term less than 2.5 or less than negative 2.5 or greater than negative 2.5, basically, what's going to happen is that if the ATP-ADP ratio is high, lots of energy around, no problems charging up, lots of things to use, charge up this ATP/ADP to a very high ratio, well, now you will favor this direction, creatine phosphate synthesis.

~17:33However, if ATP/ADP begins to fall and is lower, well, now this term is going to flip and you'll now favor ATP production. And so basically, by charging up this creatine/creatine phosphate ratio, it now creates a situation where it can buffer that ATP/ADP ratio so that when times are good, you charge up the buffer. When times are bad, you then consume the buffer in a way that allows you to protect your ATP/ADP ratio for a short time. And this is illustrated very nicely here in this slide that basically shows what happens in muscle physiology. And so this is just a theoretical curve that if you suddenly need to contract your muscle and you stop ATP production, well, ATP levels will fall. That ATP/ADP ratio more correctly will fall rapidly within the cell. Creatine phosphate will then now kick in to protect that ATP/ADP ratio as you can ramp up either anaerobic or aerobic metabolism to now buffer that ATP/ADP ratio and keep that muscle functioning. Now I want to make one point because oftentimes biochemistry textbooks will refer to things like ATP or creatine phosphate as, quote unquote, "high energy compounds." And of course, I guess they are high energy because they have these labile phosphate-nitrogen or phosphate-oxygen bonds. And because they're labile, hydrolysis is favorable. And to a certain extent, it's that hydrolysis that is providing energy. But remember it's not the metabolite or the bond in isolation that's really providing the energy. It's these ratios of ATP to ADP, creatine phosphate to creatine, that allow you to couple, for all the reasons we've spent now a lecture and a half discussing, two other reactions that really provides biological energy. All right. Now we're ready to discuss how it is that we can use glucose oxidation, nutrient catabolism, to keep ATP/ADP high, that is in a useful range, where it can allow cells to do otherwise thermodynamically unfavorable things. So how could we make this happen? Well, just like this example with creatine and creatine phosphate, if we want to use nutrient catabolism to do the same thing, we need to couple reactions of glucose oxidation, nutrient catabolism, that will allow us to synthesize ATP, ADP to ATP, and have that synthesis be favorable despite the high ATP/ADP ratio in cells. So let's think about it. So what is this? Nutrient catabolism. What did we talk about last time? Oxidizing glucose to carbon and CO2. So here's our reaction for that. So here's carbohydrate, glucose CH2O6 plus six O2 goes to six CO2 plus six H2O. That's the chemical formula for burning wood. So delta G0 prime for this is minus 686 kcals per mole. Quite a negative number. Equilibrium greatly favors the completely burned state, oxidized state, of that carbohydrate. And so you can imagine that this equilibrium is so favorable to the right, if we couple this reaction to ATP synthesis, you're clearly going to have plenty of energy there in order to maintain that ATP/ADP ratio in the right range to be compatible with cells using it to do other things. All right. So how do we actually do that? Well, if we do it, we have to follow the same rules of thermodynamics and do the same things that we did before. Basically, we have to do this reaction in a way that we can draw out a similar type of equation as we did for creatine and creatine phosphate. And at the highest level, that is how nutrient catabolism will allow us to keep ATP/ADP ratio high even as ATP hydrolysis is constantly fueling all these other unfavorable processes in the cell.

~20:55Now if we did this all in one step, our burning wood example, lots of heat is generated. That's too much energy. Not so good for biology. But rather what biology does, as we alluded to last time, is that because what we talked about in the beginning, that all of the things that we discussed are true for individual reactions or many, many reactions, basically, if we turn A into B and we do it in one step, like burning wood, or we do it in 500 steps, the thermodynamic consequences are exactly the same. And so what we need to do is do stepwise catabolism of glucose and take advantage of the fact that that delta G change is the same whether it's one step or many steps and in that stepwise fashion build in reactions where we create intermediate chemistries that allow us to do things like we did with creatine and creatine phosphate such that we can favor ATP synthesis when ATP/ADP ratio is in the physiological range. And in essence, this is the magic of how biology does this and basically couples nutrient catabolism in order to provide energy. And so the pathway that we're going to discuss today and in the next lecture is something called glycolysis, which simply put is glucose lysis. It's the stepwise chemical oxidation of glucose, which we know from our burning wood example is favorable. And we're going to use this pathway that basically will be a pathway to allow the stepwise lysis of glucose, a favorable reaction, to individual steps that set it up in a way such that ADP to ATP synthesis is favored despite a physiological ATP/ADP ratio. Now I want to point out that this really illustrates, back to that high energy compound thing, that ATP is not the only energy currency in the cell. In fact, glucose is much more energy for a cell than ATP. You get much more energy released from burning wood than you get from hydrolyzing ATP. And in essence, this is why we use glucose-- why nature has used glucose, and other molecules like it, as an energy storage device, because here is a relatively stable molecule, can sit around for a long time, and you can burn it when you need it to keep your ATP-ADP ratio high. All right. So, at the level of reactions and pathways now, how can we use glucose oxidation and couple it to drive ATP synthesis? Well, hopefully it's very clear that we have to basically set up a situation like this creatinecreatine-phosphate system that we already discussed. And so I want to start by discussing, what are the reactions in glycolysis that ultimately behave like this and allow this favorable ADP-to-ATP synthesis? And then we will go ahead and consider how we can make those reactions work in the context of an entire pathway that allows glucose oxidation. All right. Now, like creatine, phosphate, and creatine, both of the steps in glycolysis that favor ATP synthesis involve intermediates where you have phosphate on some molecule, plus ADP goes to ATP plus the phosphate coming off that molecule. Now, if we go to our creatine-creatine-phosphate system, obviously this equilibrium needs to lie to the right. And so the first one of these is as follows. It involves this molecule, phosphoenolpyruvate, also referred to as PEP. You will see throughout the course that we love to give these little acronyms to our metabolites because it's hard to constantly say and write out phosphoenolpyruvate. Much easier to say PEP. So phosphoenolpyruvate plus ADP goes to ATP. Plus, if I take the phosphate off of there, I get this molecule. This molecule is enolpyruvate. Enolpyruvate can be rearranged as follows. To make this molecule a ketone pyruvate.

~24:24You'll note-- hopefully remember from your organic chemistry classes that ketones like pyruvate can exist in two forms. They can exist in the ketone form or the enol form, rearranging via the chemistry that I've shown. The keto form is greatly favored over the enol form. However, by having a phosphate trap pyruvate the enol form-phosphoenolpyruvate, when you remove the phosphate and it's wanting to exist in the keto form, greatly enhances the energetics of this-- basically makes the reaction, the equilibrium want to lie very far to the right. And by the way of chemical tricks like this and others like it, you will see used over and over in biology. In fact, this is illustrated, if we asked what's delta G0 prime for this reaction, it's minus 15 kcals per mole. That tells us that the equilibrium of this reaction lies to the right. In fact, it lies far to the right, and in fact, this is-- you may remember ATP hydrolysis is negative 7.5 kcals per mole. So lies twice as far to the right as ATP hydrolysis does. And in fact, this minus 15 kcals per mole is the most favorable, at least in terms of individual reactions that at least I know of in metabolism. All right. Well, if this equilibrium for this is PEP to pyruvate, this here is delta G0 prime for ATP-ADP. So if we want to know what's delta G0 prime for the entire reaction, well, if PEP to pyruvate is minus 15, ATP to ADP is minus 7.5. Well here, we're going in the opposite direction, so it'd be plus 7.5. So it's minus 15 plus 7.5, means that for the entire reaction, this is minus 7.5 kcals per mole.

~26:00Means that this is favorable, equilibrium lies to the right. If we drew out-- I'm not going to do it again because I've done it a lot, but if we redrew out this equation, replacing pyruvate and PEP in the correct places, there for creatine, phosphate, and creatine, you would see that there's lots of conditions within cells that will favor ATP synthesis even when ATP-ADP ratio is high. All right. This reaction is catalyzed by an enzyme called pyruvate kinase. And pyruvate kinase is a classic example of a, quote-unquote, "irreversible reaction." However, I want to point out that the enzyme pyruvate kinase is actually named for the reverse reaction that's supposedly irreversible. Pyruvate kinase. If you take pyruvate and you act on the kinase as you learn from Professor Yaffe, that phosphorylates something. You phosphorylate pyruvate, you get phosphoenolpyruvate pyruvate kinase. And so remember that absolutely no reaction is reversible. It's only reversed-- what they mean by irreversible when they say pyruvate kinase step in glycolysis is irreversible, is that it is effectively irreversible under the conditions that are compatible with life that exist in cells. Of course, if you have very low ADP and very high ATP, you can come up with a way to net synthesize PEP. You just can't drive the reverse reaction under the ATP-ADP and the PEP pyruvate ratios that physiologically exist in cells. However, this is set up in a way such that you can favor ADP-to-ATP synthesis by converting PEP to pyruvate in a pathway, as we will see happens in glycolysis. All right. Now, the second reaction that I want to-- that allows ATP synthesis in glycolysis is catalyzed by an enzyme called phosphoglycerate kinase. And this carries out the following reaction. OK. So this molecule is 1,3-Bisphosphoglycerate, abbreviated 1,3-BPG. And basically the phosphate is transferred from here to ADP, which gives you this molecule, 3-phosphoglycerate or 3-PG.

~27:44So this reaction, the delta G0 prime for the entire coupled reaction, both steps of the reaction, is minus 4.5 kcals per mole. Tells you about the equilibrium constant. Equilibrium lies to the right, doesn't lie as far to the right as the PEP plus ADP goes to ATP plus pyruvate reaction, but nonetheless, is one where there's lots of conditions that are favorable in cells to maintain a high ATP-ADP ratio, although we will see later that this reaction actually runs quite close to equilibrium in cells, so there's actually conditions where it can go in the opposite direction as well, and we'll get to that later in the course. I want to point out one thing about this, one thing that makes this reaction favorable, as you notice here that this is a phosphate on an acid group, a so-called acid anhydride. And this is a pretty good phosphate donor. And so you want to lose that phosphate, which is one of the reasons why this reaction is favorable for ATP synthesis even at high ATP-ADP ratios. All right. So these are the two reactions in glycolysis that can favor ATP synthesis at a high ATP-ADP ratio just like creatine and creatine phosphate. And so hopefully now it's become apparent to you that if during our pathway of glucose catabolism we can build a pathway that couples what we already know is favorable, glucose oxidation, to generate these intermediates, PEP and 1,3-Bisphosphoglycerate, lacerate we can then build a pathway where those reactions can make synthesis of ATP favorable even though there's a high ATP-ADP ratio in cells, and in fact, maintain that high ATP-ADP ratio in cells. And in essence, this is the logic or the goal, if you will, of glycolysis and why it's important to provide energy to cells. OK. All right. Now, if we're going to make a pathway where glucose oxidation is coupled to making PEP and 1,3Bisphosphoglycerate, it's obvious we need to add phosphate to the system. There's no phosphate molecule on glucose. Now already mentioned earlier-- and I just erased it-- that to trap glucose in the cell, the first step in glycolysis, you add phosphate. But that phosphate donor is ATP. And so ATP being the phosphate donor doesn't help us here. If we're going to use phosphate transfer here, we need to have phosphate added into the system if this is going to work. We can't have ATP as our phosphate donor. You also saw lots of reactions from Professor Yaffe that involved phosphate transfer reactions. But again, ATP-- or GTP is the phosphate donor. And so for this system to work, we need a way to get inorganic phosphate onto a carbohydrate or we can't make things like PEP or 1,3-Bisphosphoglycerate that will allow ATP synthesis. Now we already saw earlier that additional phosphate to glucose is not favored. Remember, delta G0 prime of phosphorylating glucose was plus 3.3 kcals per mole. And it turns out that says equilibrium lies to the wrong side, so we can't net do that without ATP or energy input. And it turns out this is true for other sugars as well. And so you need energy input to add phosphate to the sugars. Plenty of energy input from glucose oxidation, but we need a reaction where phosphate addition is coupled to another reaction that makes it favorable, and we have to do this without making ATP. And again, it illustrates, again, the biological energy is not just about ATP. At a deeper level It's something different. And so to really discuss this and understand it, I need to introduce two concepts that are going to come up again over and over again in this class and across metabolism. The first of these is a very high-level bioenergetic concept, although it's really an energetic concept, and it comes back to what we already talked about.

~31:19So, we know burning wood is favorable. The CN H2O N plus oxygen going to CO2 plus water. That's burning wood, lots of energy released. Drew it up there for a single-glucose molecule and what the energy release the delta G0 prime is for that reaction. Now I want to point out chemically what is going on in this reaction. Chemically what is happening for this to occur is that electrons from the carbon are being moved to the oxygen. You are changing the oxidation state of carbon and of oxygen in order to carry out this reaction. Oxygen is a better electron acceptor than carbon, and so transferring those electrons from the carbon to the oxygen is favorable. That is, in essence, what is going on in this very favorable reaction to burn wood. Let me illustrate that a bit more explicitly here. So the carbon-- most of the carbons in carbohydrates are alcohols. So here, you can call this our carbohydrate, with the exception of the ketone or the aldehyde if it's a ketose or an aldose. All the other carbons in the sugar are alcohols. All the other bonds there. And so if we oxidize that carbon-- that is, if we remove electrons from the carbon, how can we do that? So this here generates this H- hydride ion. This is, in essence, two electrons that we are removing from the carbon. That gives us a ketone. So if we oxidize the alcohol and we remove two electrons, we get the ketone. We could oxidize this ketone further. That will give us the carboxylic acid. If we oxidize it even further, now we get CO2. So, moving in this direction, this is oxidation. Transferring electrons from the carbon to the oxygen to go in this oxidation direction, that is glucose oxidation is what's happening when we're burning wood. It's what happens when we burn glucose. Energy is released. This is largely what catabolism is about in biological systems. Now then reasons, then, that if we're going to move in the opposite direction, so-called reduction, that is going in this direction. Reduction. That's largely energy storage or anabolism in metabolism. We are storing energy that we can burn later. And of course, what's the more extreme version of this? Well, what if we add two more electrons to the carbon going in this direction? Well, what do we end up with? Well, now we end up with the saturated carbon. more reduced than the alcohol is the fully saturated carbon. You will see later that this is what fat is. You saw that in some of the earlier lectures. It's also what gasoline is. What's gasoline? It's fully saturated hydrocarbons that we then burn to CO2 and derive energy. Just like burning wood, burning gasoline all in one step, oxidizing the gasoline gives you energy. There's more energy in gasoline. You can release more energy per weight from gasoline, from fully-saturated hydrocarbons than you can from wood. Same thing happening here. What has more calories in your food? Everyone knows fat has more calories per weight than sugar. It's because the more reduced our carbon is, the more energy it stores, and as we oxidize that carbon, that is what releases the energy. And so energy transduction in biological systems-- and actually, in non-biological systems, as I just illustrated with the wood and gasoline example, but definitely in biological systems, is largely about oxidation and reduction reactions, and you are going to see this coming up over and over again.

~34:34Now, based on what I just said, the real magic, if you will, of glycolysis can be further refined to say that what glycolysis does is it couples the favorable oxidation of carbon with reactions that allow phosphate additions and production of intermediates, like PEP and 1,3-Bisphosphoglycerate, where synthesis of ATP is favored despite the high ATP-ADP ratio in cells. If you think about it, that process then allows you to keep that ATP-ADP ratio in the range where it can then be useful for all the reasons we've been talking about now for two lectures. Allows the cell to couple that ATP-ADP ratio, that-- in ATP hydrolysis to otherwise carry out otherwise unfavorable reactions. OK. So, based on that, arguably the key, or at least a very important reaction in glycolysis, is the oxidation step which happens to add the phosphate and also happens to make 1,3-Bisphosphoglycerate, which we saw earlier can be used to drive ATP synthesis. And so that reaction is the following. So this molecule hopefully looks familiar to you from the carbohydrate lectures. This is D-glyceraldehyde 3phosphate. I will abbreviate it frequently is G-3P for glyceraldehyde 3-phosphate, but it's D-glyceraldehyde 3phosphate. Remember, it's a D, sugar, because the OH group is pointing to the right as we drew it this way. It's also an aldose because it's a aldehyde sugar, and it's phosphorylated on the 3 position-- remember the way we numbered sugars was starting at the carbonyl-- 1, 2, 3. This is carbon-3, phosphorylation on the 3 position. Glyceraldehyde 3-phosphate. OK. So, this step generates 1,3-Bisphosphoglycerate. Well, to do this, there's actually two things that happened here. First we had to add the phosphate to this. It says two phosphates on it. bisphosphoglycerate, this has a single phosphate. So inorganic phosphate has to be added to generate that molecule. By the way, adding that phosphate delta G0 prime is positive plus 1.5 kcals per mole. That means even equilibrium, this reaction would lie to the left, of course, because delta G0 prime is positive. However, remember the next step downstream of this is the one shown over here where we do ATP synthesis. That step is very favorable. So this is an example where metabolism has coupled a very favorable step to a step that's less favorable. That can keep the 1,3-BPG levels low and pull the entire system forward, the metabolic trick we discussed in the last lecture. OK. You'll note the other thing that's happening here, other than adding that phosphate group, is that we've changed this aldehyde to a carboxylic acid. That is an oxidation reaction. Ketone to the acid is an oxidation reaction. I will illustrate over here this so you can see why it's an oxidation reaction. So here's our aldehyde. If we take electrons from the carbon and that generates this hydride ion to electrons, what are we left with? Well, we're left with this intermediate that will never exist with a positive charge on the carbon. Water, which, of course, is abundant in biological systems. And then come-- OK. And it basically illustrates that as I go from the aldehyde to the acid, of course, I make a proton and a hydride ion, but really, I have changed the oxidation state of the carbon from the ketone to the acid state. Two electrons are lost there, is shown as that hydride ion. OK. So, what does that mean? Well, that means that we have to maintain that balance in this reaction as well. So water is going to have to come in and a proton will be produced. But the key thing is we also generate two electrons, this hydride ion that have to go somewhere. Now, finding a place for those two electrons to go is what brings up the second high-level concept that I now need to spend some time discussing.

~38:08And this second high-level concept that I need to introduce is the role of cofactors, and cofactors also is effectively the role of vitamins and what role that these things play in metabolism. Most vitamins are used to support metabolic reactions, and we're going to learn all about what many of the vitamins that you guys are familiar with reading the side of your cereal box actually do throughout the course.

~38:31Now cofactors, as I said, often involve vitamins, are molecules that provide useful chemical groups that facilitate the reaction chemistries that are needed in metabolism. So they provide some useful chemical groups to facilitate some of the chemistries that we need to carry out metabolic reactions. They are, in general, in small non-stoichiometric quantities in order to carry out this function. What do I mean by that? Meaning that you can imagine that the cell will convert lots of glyceraldehyde 3-phosphate to 1,3Bisphosphoglycerate, but the enzyme, you don't need one enzyme for every time you convert that reaction. The same enzyme can do it over and over and over again. Well, it turns out, cofactors are much more like enzymes in that respect. They need to be recycled in a way that they can catalyze the turnover of reaction many, many, many times, and it'll be much clearer what I mean by this when we get into some of the details. Now, the vitamin concept comes from the fact that often these cofactors or parts of the cofactors are not made by animals, not made by humans. If they're not made by us, but we need them to do our metabolism, well now we have to get them from the diet, and this is really the concept of vitamins. These are small things that we have to get in quantities from the diet, and it turns out they're very important to help us carry out the metabolism we do. As an aside, the vitamin industry is a very big business. Lots of unfounded claims out there. I'm not going to get into-- that can get very political. I'm just going to say that just know that more is not always better, and the goal for this class is really to understand what some of these vitamins actually do. OK. Now, the relevant cofactor for the reaction that we're talking about is a cofactor called nicotinamide adenine dinucleotide, which is always abbreviated NAD or NAD+. This stands for nicotinamide adenine dinucleotide. You see why we want to abbreviate that NAD. And basically what nicotinamide adenine dinucleotide is is it's a factor that can solve our problem of where to put these electrons in this oxidation reaction. That is, you can accept two electrons from a reaction like this one here and cycle to another form of the cofactor called NADH. All right. What is nicotinamide adenine dinucleotide? Well first, let's draw what it looks like. OK. Sorry it's squeeze down here a little bit. So this here is adenine. This group up here is nicotinamide. So if you look at this, this is a dinucleotide. And so down here, this, of course, is, from here down, is AMP. That phosphate is now over here. This is a separate nucleotide, where rather than having one of the AGCTU bases that you're used to from RNA and DNA, nicotinamide is the base. And so it's this AMP plus this nicotinamide base is a monophosphate, or a nicotinamide adenine dinucleotide. This, the nicotinamide group, is derived from a vitamin called niacin. And so from the side of your cereal box, this is what niacin does. It allows you to generate the nicotinamide group that's part of this cofactor, nicotinamide adenine dinucleotide. Now, nicotinamide adenine dinucleotide is useful because of what that nicotinamide group can do.

~41:36And so I'm not going to draw out the whole molecule, but I'll show you. So NAD+, which is the oxidized form of nicotinamide adenine dinucleotide, is as I drew it up there. So this here would be attached to the rest of the dinucleotide. So this is the oxidized form. I can have-- here's my hydride ion, which, remember, is two electrons. If I carry out that chemistry to add those two electrons to the nicotinamide group, you now end up with the reduced form of nicotinamide adenine dinucleotide NADH. So the difference between NAD+ and NADH, it's the H, but it's really the two electrons that were added in adding that H. So it's oxidized form and reduced form. So remember, I want to stress this again, at least in biology we can't create or destroy matter. You can do that in nuclear physics, but we can't do that in biology. And so that means that if we do an electron transfer like we did in this reaction where we oxidized the aldehyde in glyceraldehyde 3-phosphate to the acid in 1,3Bisphosphoglycerate, those electrons have to be accounted for. And so if one molecule is oxidized, another one has to be reduced. So redox reactions always-- that is, redox, oxidation-reduction reactions, always have to happen in pairs. And so over there we have the aldehyde in glyceraldehyde 3-phosphate is oxidized to form the acid in 1,3-Bisphosphoglycerate. As part of that reaction, we can couple it such that the nicotinamide group, which is oxidized in NAD, can be reduced to the nicotinamide group that is found in NADH. So that means we can draw this in here, is that having that cofactor coupled to this reaction now allows us to fix the redox problem of this reaction. Now this reaction here is catalyzed by an enzyme called glycerol 3-phosphate dehydrogenase. I'll draw that out in the second. Often abbreviated GAPDH, the loading control in all of your western blots, is the enzyme GAPDH, glyceraldehyde phosphate dehydrogenase. That is the enzyme that carries out that reaction. Now notice, the name fits. It's a dehydrogenase, it's removing a hydrogen, removing two electrons as a hydride ion, but it's really-- what's important here is the electrons moving, not the hydrogen itself. So in that way it's a little bit misleading. And you will see that in general, dehydrogenases are classes of enzymes that catalyze oxidation-reduction reactions, and as a result, often involve NAD/NADH as cofactors because they facilitate those electron transfer reactions. OK. So now we're able to describe how GAPDH works in great detail. And so the chemistry of GAPDH works as follows. And so in the active site of GAPDH, there's a cystine residue. That cystine residue has a sulfur shown here. Also bound near the active site is in NAD molecule to catalyze the reaction shown. That NAD has to be in the oxidized state, so it's NAD+. And so glyceraldehyde 3-phosphate binds in the active site. There's glyceraldehyde 3-phosphate. By the way, I will show you how many reactions in metabolism work chemically. Obviously we don't have time in this class, an overview class to go into all the great chemical details of what's going on and exactly how everything works, but I'll at least try to give you a flavor of some of the chemistry that happens throughout the course. All right. So once you have, then-- OK. So now you have this intermediate bound into the enzyme active site. You can now carry out the oxidation of that carbon. So these are two electrons being removed as a hydride ion. They can be transferred to NAD in the active site. That generates NADH by the exact chemistry that I just showed you. OK.

~45:03That NADH now needs to be exchanged out or the enzyme will not be able to catalyze the next series of reactions, and that NADH will then go somewhere else, and of course, has to be cycled back to NAD, but that doesn't happen in this reaction, per se. OK. Next, this here, you'll notice, is that by the way this chemistry worked, we formed this thioester bond in the active site. This is another thing that you will see over and over again in metabolism. This sulfur in the thioester bond is a good leaving group, and so it's really setting up a situation that will now help with the phosphate addition. So here, I'll draw out inorganic phosphate. And that, of course, will give us the identical enzyme active site that we started with plus 1,3-Bisphosphoglycerate. And that 1,3-Bisphosphoglycerate can now be coupled to ATP synthesis to make 3-phosphoglycerate. And so you can see, in essence, what this reaction has done is that it has coupled oxidation of the aldehyde to the acid, the aldehyde in 3-phosphate to the acid in 1,3-Bisphosphoglycerate, which can then do phosphorylation of ADP to make ATP. So in essence, this is oxidative phosphorylation. Now I realize in high school or in other classes, oxidative phosphorylation means something different to you, and of course, we will discuss the process that is called oxidative phosphorylation in great detail later in this course. But I want to point out that the chemistry that's actually happening here in glycolysis is also oxidative phosphorylation. It's basically how you coupled this favorable carbon oxidation that's occurring at this GAPDH step in a way that allows you to synthesize ATP at a high ATP-ADP ratio, which in the end will allow cells to do otherwise unfavorable things. OK. Now we're ready to discuss these reactions, the GAPDH reaction, the pyruvate kinase reaction, the phosphoglycerate kinase reaction, and how they fit into glycolysis as a metabolic pathway in a way that allows cells to use glucose metabolism-- that is, the breakdown of glucose, the burning of glucose as a way to get energy. And that pathway is, of course, glycolysis or glucose lysis that we mentioned earlier. Glycolysis is a very ancient and ubiquitous pathway. It's used by essentially all life on Earth and probably has been for at least 2 to 3 billion years. Glycolysis, I guess, as a pathway was, quote-unquote, "discovered" mid-19th century by Louis Pasteur, and Louis Pasteur, of course, described life, microorganisms as being responsible for the process of fermentation. That is, conversion of glucose into alcohol, something many college students are interested in, as well as other organic acids. And of course, fermentation to make alcohol or to store other types of organic acids has been used in food preservation for centuries, which is really why Pasteur's discoveries were so impactful at the time. Now even though Pasteur discovered this, he didn't really understand chemically how it worked, and it actually took decades to figure that out. And when it was finally figured out and described as the pathway called glycolysis, this was done by two chemists, two chemists in Germany, Embden and Meyerhof. And basically what they did is they pieced together several enzymatic or chemical activities that have been purified from cell lysates mostly from yeast and used those to build a pathway that described chemically how you could start with glucose and chemically break down that glucose in a way that made sense in this fermentation pathway, and that is why glycolysis is sometimes called the Embden-Meyerhof pathway.

~48:20Now, glycolysis does not completely convert glucose to CO2. Instead, what glycolysis does is it converts glucose-so I will draw here in the pyranose form. It converts glucose by a number of steps into two molecules of the organic acid pyruvate, which, of course, was the product of that pyruvate kinase reaction that we showed earlier. Note that the fermentation that Louis Pasteur described was further metabolism of that pyruvate into something else. In the case of yeast, it was ethanol or ethyl alcohol, or something else. The fermentative product in mammals is this organic acid lactate. And what we will see next time is that this fermentation of pyruvate into one of these other molecules ends up being an alternative to the further oxidation of pyruvate to CO2 which requires oxygen, and the reason oxidizing pyruvate to CO2 requires oxygen is because ultimately we have to regenerate that NADH we made in glycolysis to allow that cofactor to cycle and glycolysis to keep moving. Fermentation gives us an alternative way to do this, which we'll discuss in great detail in the next lecture. But first I want to focus on glycolysis and what we can describe next time as really how we can build a pathway to turn glucose into pyruvate. This glycolysis, this Embden-Meyerhof pathway, and some of the details of that pathway are shown here on this slide. And this is where we'll start off the next lecture, discussing how each of these steps in glycolysis function in a way that allow a pathway to generate phosphoenolpyruvate, 1,3-Bisphosphoglycerate, incorporate this GAPDH reaction, and do so in a way that's overall energetically favorable to allow the cell to couple this oxidation of glucose to keeping ATP-ADP ratio in the right range to support other unfavorable functions in the cell. OK, thanks.

2.3获能期收官（第 8–10 步）：2,3-BPG 乒乓变位、脱水造势与 PEP 互变异构驱动的终极能量爆发

一、第 8 步与第 9 步：为什么 3-PG 不能直接造 ATP，而要先移位再脱水？

经过第 7 步收回 2 ATP 后，我们手里剩下两分子 3-磷酸甘油酸（3-PG），上面还挂着准备期装上去的那两个磷酸基团。细胞能不能偷个懒，直接把 3-PG 上的磷酸转给 ADP 合成 ATP 呢？绝对不行！因为 3-PG 是一个普通的醇羟基磷酸单酯（低能磷酸键），其水解标准自由能只有 Δ G'^° = -12.6 kJ/mol（-3.0 kcal/mol），远远不够支付合成 ATP 所需的 +30.5 kJ/mol！

怎样才能把一个平平无奇的“低能醇磷酸酯”，点石成金变成一个“超高能磷酸化合物”呢？大自然的化学智慧是：先把磷酸从 C3 挪到 C2（第 8 步），再脱去一分子水形成双键（第 9 步），把低能磷酸酯改造成被困在烯醇式里的“高能烯醇磷酸酯（PEP）”！

第 8 步：磷酸基团分子内移位（以 2,3-BPG 为引物的乒乓机制）

3-磷酸甘油酸（3-PG） ⇌(磷酸甘油酸变位酶（PGAM）｜需微量 2,3-BPG 作引物) 2-磷酸甘油酸（2-PG） (Δ G'^° = +4.4 kJ/mol)

• Vander Heiden 课堂拆解的“乒乓接力”机制：你在初学时可能会以为，磷酸甘油酸变位酶（Phosphoglycerate mutase, PGAM） 是把同一个 3-PG 分子 C3 上的那个磷酸直接揪下来按到它自己的 C2 上。Vander Heiden 教授在 L21 课堂上特意用彩色粉笔演示了真相：根本不是同一个磷酸在分子内搬家，而是酶活性中心的组氨酸（His）事先挂着一个磷酸（E-His-P），玩了一场“先给一个、再拿回一个”的乒乓游戏！

1. 引物激活（Priming）：在反应开始前，必须有微量的辅因子 2,3-二磷酸甘油酸（2,3-BPG） 充当引物，先把一个磷酸基团交给酶活性中心的组氨酸残基，使酶处于磷酸化激活态（E-His-P）。
2. 催化循环：当底物 3-PG 进入活性中心后，E-His-P 把自己身上的磷酸加到 3-PG 的 C2-OH 上，形成短暂的 酶结合态 2,3-BPG 中间物；紧接着，该中间物把原来 C3 位上的那个磷酸反手交还给酶的 His 残基，重新恢复 E-His-P 活性态，同时释放出产物 2-磷酸甘油酸（2-PG）！

第 9 步：2-磷酸甘油酸脱水生成 PEP（分子内能量重排）

2-磷酸甘油酸（2-PG） ⇌(烯醇化酶（Enolase）, Mg²⁺/Mn²⁺｜F^- 强烈抑制) 磷酸烯醇式丙酮酸（PEP） + H_2O (Δ G'^° = +7.5 kJ/mol)

• 从低能到超高能的质变：由 烯醇化酶（Enolase，需 Mg²⁺ 或 Mn²⁺，被氟化物 F^- 强烈抑制） 催化 2-PG 发生 β-消除脱水反应。请注意一个极具震撼力的热力学对比：脱水反应本身的 Δ G'^° 只有 +7.5 kJ/mol（标准态可逆），但底物 2-PG 的磷酸酯键水解 Δ G'^° 仅为 -17.6 kJ/mol（低能磷酸键），而脱水产物 磷酸烯醇式丙酮酸（Phosphoenolpyruvate, PEP） 的磷酸键水解 Δ G'^° 竟然暴增到了惊人的 -61.9 kJ/mol（课堂取 -15 kcal/mol）——整整是 ATP 水解自由能的两倍，稳居生物体内所有已知高能磷酸化合物榜首 2013·名解！

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二、第 10 步：丙酮酸激酶与烯醇-酮式互变异构驱动的第二次底物水平磷酸化

手握全代谢界能量最高的“王炸底物” PEP，糖酵解迎来了收官的第 10 步——在 丙酮酸激酶（Pyruvate kinase, PK，需 Mg²⁺ 和 K⁺） 催化下，将高能磷酸基团转移给 ADP，完成 第二次底物水平磷酸化：

磷酸烯醇式丙酮酸（PEP） + ADP + H⁺ ──(丙酮酸激酶（PK）, Mg²⁺, K⁺)──► 丙酮酸（Pyruvate） + ATP (Δ G'^° = -31.4 kJ/mol 或 -7.5 kcal/mol)

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三、糖酵解（EMP 途径）十步反应总表与全方位能量精算

为了让你在考场上无论遇到填空、单选还是大型能量计算题都能秒杀，我们将 EMP 途径全部 10 步反应及能量账单浓缩于下表：

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🎧 MIT 7.05 课堂原声英文实录对照（本节关联 2 讲 · 完整逐段实录）点击展开 / 收起 ▾

🎬 MIT 7.05 Lec 20 · Bioenergetics & Glycolysis I (Prof. Vander Heiden)⏱ ~50:00

~00:00MITOCW | 20. Bioenergetics/Intro Pathways/Glycolysis I PROFESSOR:

~00:02Today we're going to discuss bioenergetics, continue that discussion and use that as a transition into also beginning to see how that applies to understanding glycolysis, the pathway that allows glucose breakdown. So just as a reminder of a couple of the points that we discussed last time, so last time we discussed what makes reactions favorable. And so remember, you could have any reaction, A to B-- A to B could be a single step. A to B could be an entire pathway, all steps in the pathway, that whether or not that reaction or pathway happens is determined by thermodynamics. It's not determined by enzymes. Enzymes are important, of course, to control the rate of reactions so they may allow things to happen that otherwise wouldn't happen. But whether the reaction happens or not is determined by free energy. And so free energy, delta G, remember we discussed is the following relationship. So it's related to this constant delta G0 prime, which is related to the equilibrium constant for any pair of products and reactants as well as this formula rt times the log of the products over the reactants. And so remember this means that whether a reaction happens depends on the equilibrium constant, but it also depends on the actual conditions present. That is how much substrate and how much product is there. And so what this means is that biology can come up with ways by keeping the product concentrations low to do things that may move in the opposite direction of what you would predict from equilibrium. And also by coupling these less favorable reactions to more favorable reactions, you can also allow biology to do unfavorable stuff. And this is, of course, where the role of ATP comes in. And we discussed this at the end of last time as well. Of course, what's important then becomes the ratio of ATP to ADP. And to illustrate that, we use this example. We use the-- remember ATP hydrolysis has a delta G0 prime of minus 7.5 kcals per mole. That, of course, tells you this is about the equilibrium constant. That tells us the equilibrium lies to the right. We can couple it to this first reaction in glycolysis, which traps glucose in the cell by adding a phosphate group. Delta G0 prime for this is positive 3.3 kcals per mole. This positive number tells you that the equilibrium lies towards the left. But when we couple these two reactions together, we add these two numbers together, and now we get a delta G0 prime of minus 4.2 kcals per mole, which are the coupled reaction. The equilibrium lies now again to the right. And that's how we can use ATP hydrolysis in order to carry out this unfavorable process, trapping the glucose in the cell.

~02:38Now how much glucose will be trapped? Well, of course, that will come from this reaction up here. And that's because whether or not a reaction occurs or not of course depends not on the equilibrium constant but on the actual conditions and the delta G of that reaction.

~02:54And so we can just plug that in for this system. And so we see delta G0 prime minus 4.2 kcals per mole. That says equilibrium lies to the right. But how far really depends on the conditions. And that's defined by this ratio of products over reactants. And you can imagine that this term here, as long as it's less than 4.2, you will favor glucose trapping inside the cell, whereas, of course, if it's greater than 4.2, then you'd favor-- the opposite direction would be favorable. Now this is really critical because this really explains how it is that ATP actually works to help cells do various reactions. And so if an ATP-ADP ratio exists such that it can make delta G less than 0 for the reaction that it's coupled to, that is how it provides the energy that then allows unfavorable reactions to occur. And so please do not make the common mistake that many, many biologists make when they state, oh, ATP equals energy. There's a lot of ATP. The cell must have a lot of energy. Remember it's the ratio of ATP to ADP that provides the energy. And so that means ATP can be sky high, but if ADP is equally high, there's no energy there. Consequently, ATP can be very low. But if ADP is extremely low, now you still have a high ATP to ADP ratio. And that is what provides the energy. And that's because this occurs regardless of concentration. It's because it's this ratio of ATP to ADP that ultimately says how much energy is there for the ATP to drive a reaction. In other words, delta G, or whatever the reaction is coupled to, is going to be proportional to the ratio of ADP to ATP. Great. Now how much ATP do you need then? What ratio do you need to drive a reaction? Well, it's hard to say that in absolute terms because it's going to be defined by how that ratio is coupled to the ratio of whatever other reactants and products. It also tells you, remember, as we talked about before, we can switch the directions of these reactions and we just flip the signs. The directions here are arbitrary. And so that means it also tells us how much energy we need to put in if we're going to charge up an ATP-ADP ratio. In other words, if we want to synthesize ATP, that is take ADP plus phosphate and make an ATP, well, what's this? What we just-- the exact opposite sign of that delta G0 prime is going to now equal plus 7.5 kcals per mole. And so that means that the equilibrium of this reaction is going to lie to the left just as that one lied to the right. And it's going to lie to the left by that much. And if we want to know how much energy it then takes to make ATP, well, obviously, it's not going to happen because equilibrium favors this direction. It's not spontaneous in that direction. But at various conditions, we can know by plugging in our formula delta G equals delta G0 prime plus 7.5 kcals per mole plus RT times the log of ATP. So if I have my ADP-- I'm sorry-- my ATP concentration sufficiently low relative to my ADP concentration, I can still make this reaction happen. Now that's not going to give you an ATP-ADP ratio that is actually consistent with being able to do anything else into the cell. And so but what it says is that if I am able to couple energy from nutrient metabolism to this reaction and overcome this positive 7.5 kcals per mole, that basically will allow me to now synthesize ATP and maintain an ATP-ADP ratio in the cell that allows cells to carry out other functions by the same rules that we talked about that would be unfavorable. Now there's lots of implications here for metabolism, because it says how much input you need to synthesize ATP. How much energy you need to synthesize ATP is going to depend on how charged this ratio is. And how useful that ratio is to catalyze other reactions is also going to depend on how charged that ratio is.

~06:50And in essence, this is why we cannot store energy as ATP. This is why we didn't eat ATP for breakfast and instead ate cereal that has a bunch of glucose in it, because we can use that glucose to get ATP but the ATP itself is not something that we really can burn to get energy in a sustained way. In other words, the delta G of ATP synthesis, making ATP is going to be proportional to the log of the ratio of ATP over ADP for all the reasons I described. The delta G of using ATP, ATP hydrolysis, using ATP to drive reactions is also proportional to RT times the log, in this case, of the ADP to ATP ratio. And so because you need to consume this ratio to get energy, and you need to put in energy to create this ratio, you are constant-- and how much is an exponential property-- you are constantly having to generate ATP in a way that allows you to then have a ratio that can then be consumed to fuel other reactions. And that's what's shown here on the slide. So I just graphed here what the ATP-ADP ratio is relative to a theoretical delta G that's shown here as a linear scale, here on a log scale, that basically there's this logarithmic relationship and so you need an exponential increase in energy input to drive this ratio up higher in order to, quote unquote, "store" more energy that can be used for later. And so this system is not one that you can use as an energy storage system and ultimately explains why cells have to do constant metabolism, have to constantly do some sort of metabolism to keep this ATP-ADP ratio charged for it to be in a useful range to have an ATP-ADP ratio that can be coupled to drive otherwise unfavorable reactions in our cells. So to say this another way, our cells need to constantly catabolize nutrients, burn glucose, burn some other fuel. They have to do this because they have to keep that ratio charged in a range where that can then support other cell functions, allow them to fight entropy and maintain order in biology. We alluded last time if you stop making ATP, well, what's going to happen? Well, that ATP-ADP ratio the cell has is going to fuel whatever reactions it can do until suddenly that ratio falls below the point where it's useful to drive those unfavorable reactions. In fact, if you stop ATP production, you will consume that ATP-ADP ratio in seconds. And this is why if you poison ATP production, cells die very quickly. Remember last time we mentioned you have a heart attack, blood flow stops going to a tissue. No more nutrients and oxygen. Cells die quickly. Cyanide poisoning does the same thing. Cells die quickly if you stop their ability to maintain this ATP-ADP ratio in the right range. And so remember the concentration, the absolute concentration of ATP for these reasons is not a good indicator of cell energy. The correct indicator of cell energy is the ratio of ATP to ADP. This term is sometimes referred to as the energy charge. And so that can be the ATP to ADP ratio, or oftentimes more correctly is the ATP to AMP ratio. Why the ATP to AMP ratio? Well, that's because once you understand this, you can now understand that cells have a failsafe mechanism to protect themselves if this ratio of ATP to ADP becomes too low. What is that mechanism? Well, you can carry out this reaction. Take two ATP molecules and turn them into transfer a phosphate to make an ATP plus an AMP. Why is that a failsafe mechanism? Well, if they care about this, if the ATP-ADP ratio is what maintains your ability to drive these thermodynamically unfavorable reactions, if I sacrifice the denominator by removing an ATP and get an ATP out of it, I've now at least temporarily fixed that ratio. Now this is something that obviously cannot go on forever.

~10:36But in the short term, it does allow cells to protect that ratio. And in fact, what cells actually defend is this ATPAMP ratio. And it's very, very difficult to change the ATP-AMP ratio in a cell and actually have it live. So it's really this ATP-AMP ratio that is most formally related to what would be called energy charge. But this is really why this is important is for all the reasons that I just described. All right. So hopefully, it's clear to you now why cells have to keep ATP-ADP ratio in the right range, why this is useful for them to now carry out all of these unfavorable reactions-- fight entropy, stave off death. And they can even have a failsafe here and sacrifice ADP as a way to try to maintain that as a very last resort. It also says why cells have to always do catabolism, because you always need the energy input to keep that ratio in the right range. However, you might imagine that, in real life, the messiness of biology, that there's lots of situations where the demand for ATP will suddenly increase. Well, of course, maybe you can just metabolize more sugar, do more nutrient catabolism to try to keep that ratio high. But that takes time. And so you need ways for cells to quickly respond to then allow them to mobilize all of this extra thing to-- extra fuel to do that. For example, we're walking around our ancestors on the plains somewhere and a lion appears. We don't have time to mobilize a bunch of new fuel. We have to be able to run away really quickly or the lion's going to eat us. And so cells have systems to suddenly respond to I need a lot more ATP demand to run away by having ways that one can buffer this ATP-ADP ratio, protect it in the short term, and allow cells time to ramp up nutrient metabolism in order to keep this high. All right. Well, if we wanted to create such a buffer system, how would we actually do it? Well, obviously, we have to follow all the rules of thermodynamics that we discussed. And we have to do so in a way that actually illustrates how it is that very well how metabolism works. In other words, if suddenly the ATP-ADP ratio drops, if we have a sudden drop in ATP-ADP ratio because we have increased demand, needing to run away from a lion, how could we actually create a buffer system that protects this? Well, to do it, we would need to couple ATP-ADP to some other product reactant pair, let's call them x and y, such that the ratio of y to x is coupled to the ratio of ATP to ADP. And you want it coupled in such a way that if that ratio goes down, this ratio can now protect ATP, basically favor ATP to ADP synthesis. However, when nutrients are plentiful and this ratio is really high, it then in turn can charge up this ratio so that it can buffer it later when the ATP-ADP ratio starts to fall. Now the most famous example of this actually comes from muscle physiology. And it's best described in terms of muscles and how they quickly respond to increased demand, like that running away from a lion. However, it in fact applies to many, many cells, including most non-muscle cells in animals.

~13:47All right. And I should say that it turns out that if you turn off ATP synthesis in a muscle, people have estimated that your ATP-ADP ratio will fall into a range where it is no longer useful to do anything within a matter of seconds, some claim even less than a second. And so this system ends up being very important.

~14:08Now the system that muscle uses, and many other cells do as well, is something called the creatine/creatine phosphate system. Now if you guys are weightlifters or bodybuilders out there, you probably have heard of creatinine because it's a very popular supplement that is taken for weightlifting, something I obviously don't do a lot of. But that creatine/creatine phosphate is out there. I'll describe to you what it actually does for muscle cells. And you can decide for yourself if this is actually useful as a energy or weightlifting supplement. So what is creatine/creatine phosphate? What does it look like? Well, it's basically looks like this. By the way, I will use this abbreviation often throughout this course, just a shorthand to draw a lot of these structures. So this here would be a carboxylic acid group drawn like this or like that, drawn to the carboxylic acid group just so you understand my shorthand. OK. So this here is the molecule creatine. If I put a phosphate here, this now becomes creatine phosphate. Again, just to remind you, this P with a circle over it is a phosphate group. I'll draw this particular phosphate group because it's a nitrogen-phosphate bond, a phosphoramidate bond. This is actually a bond that is more labile than the PO bonds that we're used to seeing in the phosphodiesters like ATP, et cetera. All right. So that's creatine and creatine phosphate. And if we draw out the reaction, creatine phosphate goes to creatine plus inorganic phosphate, so hydrolysis of creatine phosphate. This has a delta G0 prime of minus 10 kcals per mole. What does that mean? It's negative. Tells us about the equilibrium constant. Equilibrium lies to the right. Remember ATP to ADP hydrolysis shown over there, that hydrolysis has a delta G0 prime of minus 7.5 kcals per mole. Equilibrium also lies to the right. This number is more negative. This equilibrium lies further to the right than ATP hydrolysis does. So now what happens if we couple this reaction to the ATP hydrolysis reaction? So now we can do ATP plus creatine goes to creatine phosphate plus ADP. So we've coupled those two reactions together. So delta G0 prime, ATP to ADP is minus 7.5. Creatine phosphate to creatine in that direction is negative 10. In this direction, it would be positive 10. And so as I've drawn the reaction, this delta G0 prime will be plus 2.5 kcals per mole. 10 minus 7.5 plus 2.5, which tells us then that this equilibrium now lies to the left. However, which direction this reaction actually goes is not determined only by the equilibrium. It's really determined by delta G. So we can calculate that. So delta G equals delta G0 prime plus RT log of, as we've drawn it, creatine phosphate over creatine and ADP over ATP. So you can now imagine that this term here is the sum of those two. So it's plus 2.5. And so if this term is less than 2.5, what does that mean? Well, if it's-- I'm sorry. Less than negative 2.5, then delta G will be negative and you will favor creatine phosphate production. If this term is greater than negative 2.5, then delta G will be positive and you will favor ATP production. Why is that important? Well, if you think about the conditions, how you could make this term less than 2.5 or less than negative 2.5 or greater than negative 2.5, basically, what's going to happen is that if the ATP-ADP ratio is high, lots of energy around, no problems charging up, lots of things to use, charge up this ATP/ADP to a very high ratio, well, now you will favor this direction, creatine phosphate synthesis.

~17:33However, if ATP/ADP begins to fall and is lower, well, now this term is going to flip and you'll now favor ATP production. And so basically, by charging up this creatine/creatine phosphate ratio, it now creates a situation where it can buffer that ATP/ADP ratio so that when times are good, you charge up the buffer. When times are bad, you then consume the buffer in a way that allows you to protect your ATP/ADP ratio for a short time. And this is illustrated very nicely here in this slide that basically shows what happens in muscle physiology. And so this is just a theoretical curve that if you suddenly need to contract your muscle and you stop ATP production, well, ATP levels will fall. That ATP/ADP ratio more correctly will fall rapidly within the cell. Creatine phosphate will then now kick in to protect that ATP/ADP ratio as you can ramp up either anaerobic or aerobic metabolism to now buffer that ATP/ADP ratio and keep that muscle functioning. Now I want to make one point because oftentimes biochemistry textbooks will refer to things like ATP or creatine phosphate as, quote unquote, "high energy compounds." And of course, I guess they are high energy because they have these labile phosphate-nitrogen or phosphate-oxygen bonds. And because they're labile, hydrolysis is favorable. And to a certain extent, it's that hydrolysis that is providing energy. But remember it's not the metabolite or the bond in isolation that's really providing the energy. It's these ratios of ATP to ADP, creatine phosphate to creatine, that allow you to couple, for all the reasons we've spent now a lecture and a half discussing, two other reactions that really provides biological energy. All right. Now we're ready to discuss how it is that we can use glucose oxidation, nutrient catabolism, to keep ATP/ADP high, that is in a useful range, where it can allow cells to do otherwise thermodynamically unfavorable things. So how could we make this happen? Well, just like this example with creatine and creatine phosphate, if we want to use nutrient catabolism to do the same thing, we need to couple reactions of glucose oxidation, nutrient catabolism, that will allow us to synthesize ATP, ADP to ATP, and have that synthesis be favorable despite the high ATP/ADP ratio in cells. So let's think about it. So what is this? Nutrient catabolism. What did we talk about last time? Oxidizing glucose to carbon and CO2. So here's our reaction for that. So here's carbohydrate, glucose CH2O6 plus six O2 goes to six CO2 plus six H2O. That's the chemical formula for burning wood. So delta G0 prime for this is minus 686 kcals per mole. Quite a negative number. Equilibrium greatly favors the completely burned state, oxidized state, of that carbohydrate. And so you can imagine that this equilibrium is so favorable to the right, if we couple this reaction to ATP synthesis, you're clearly going to have plenty of energy there in order to maintain that ATP/ADP ratio in the right range to be compatible with cells using it to do other things. All right. So how do we actually do that? Well, if we do it, we have to follow the same rules of thermodynamics and do the same things that we did before. Basically, we have to do this reaction in a way that we can draw out a similar type of equation as we did for creatine and creatine phosphate. And at the highest level, that is how nutrient catabolism will allow us to keep ATP/ADP ratio high even as ATP hydrolysis is constantly fueling all these other unfavorable processes in the cell.

~20:55Now if we did this all in one step, our burning wood example, lots of heat is generated. That's too much energy. Not so good for biology. But rather what biology does, as we alluded to last time, is that because what we talked about in the beginning, that all of the things that we discussed are true for individual reactions or many, many reactions, basically, if we turn A into B and we do it in one step, like burning wood, or we do it in 500 steps, the thermodynamic consequences are exactly the same. And so what we need to do is do stepwise catabolism of glucose and take advantage of the fact that that delta G change is the same whether it's one step or many steps and in that stepwise fashion build in reactions where we create intermediate chemistries that allow us to do things like we did with creatine and creatine phosphate such that we can favor ATP synthesis when ATP/ADP ratio is in the physiological range. And in essence, this is the magic of how biology does this and basically couples nutrient catabolism in order to provide energy. And so the pathway that we're going to discuss today and in the next lecture is something called glycolysis, which simply put is glucose lysis. It's the stepwise chemical oxidation of glucose, which we know from our burning wood example is favorable. And we're going to use this pathway that basically will be a pathway to allow the stepwise lysis of glucose, a favorable reaction, to individual steps that set it up in a way such that ADP to ATP synthesis is favored despite a physiological ATP/ADP ratio. Now I want to point out that this really illustrates, back to that high energy compound thing, that ATP is not the only energy currency in the cell. In fact, glucose is much more energy for a cell than ATP. You get much more energy released from burning wood than you get from hydrolyzing ATP. And in essence, this is why we use glucose-- why nature has used glucose, and other molecules like it, as an energy storage device, because here is a relatively stable molecule, can sit around for a long time, and you can burn it when you need it to keep your ATP-ADP ratio high. All right. So, at the level of reactions and pathways now, how can we use glucose oxidation and couple it to drive ATP synthesis? Well, hopefully it's very clear that we have to basically set up a situation like this creatinecreatine-phosphate system that we already discussed. And so I want to start by discussing, what are the reactions in glycolysis that ultimately behave like this and allow this favorable ADP-to-ATP synthesis? And then we will go ahead and consider how we can make those reactions work in the context of an entire pathway that allows glucose oxidation. All right. Now, like creatine, phosphate, and creatine, both of the steps in glycolysis that favor ATP synthesis involve intermediates where you have phosphate on some molecule, plus ADP goes to ATP plus the phosphate coming off that molecule. Now, if we go to our creatine-creatine-phosphate system, obviously this equilibrium needs to lie to the right. And so the first one of these is as follows. It involves this molecule, phosphoenolpyruvate, also referred to as PEP. You will see throughout the course that we love to give these little acronyms to our metabolites because it's hard to constantly say and write out phosphoenolpyruvate. Much easier to say PEP. So phosphoenolpyruvate plus ADP goes to ATP. Plus, if I take the phosphate off of there, I get this molecule. This molecule is enolpyruvate. Enolpyruvate can be rearranged as follows. To make this molecule a ketone pyruvate.

~24:24You'll note-- hopefully remember from your organic chemistry classes that ketones like pyruvate can exist in two forms. They can exist in the ketone form or the enol form, rearranging via the chemistry that I've shown. The keto form is greatly favored over the enol form. However, by having a phosphate trap pyruvate the enol form-phosphoenolpyruvate, when you remove the phosphate and it's wanting to exist in the keto form, greatly enhances the energetics of this-- basically makes the reaction, the equilibrium want to lie very far to the right. And by the way of chemical tricks like this and others like it, you will see used over and over in biology. In fact, this is illustrated, if we asked what's delta G0 prime for this reaction, it's minus 15 kcals per mole. That tells us that the equilibrium of this reaction lies to the right. In fact, it lies far to the right, and in fact, this is-- you may remember ATP hydrolysis is negative 7.5 kcals per mole. So lies twice as far to the right as ATP hydrolysis does. And in fact, this minus 15 kcals per mole is the most favorable, at least in terms of individual reactions that at least I know of in metabolism. All right. Well, if this equilibrium for this is PEP to pyruvate, this here is delta G0 prime for ATP-ADP. So if we want to know what's delta G0 prime for the entire reaction, well, if PEP to pyruvate is minus 15, ATP to ADP is minus 7.5. Well here, we're going in the opposite direction, so it'd be plus 7.5. So it's minus 15 plus 7.5, means that for the entire reaction, this is minus 7.5 kcals per mole.

~26:00Means that this is favorable, equilibrium lies to the right. If we drew out-- I'm not going to do it again because I've done it a lot, but if we redrew out this equation, replacing pyruvate and PEP in the correct places, there for creatine, phosphate, and creatine, you would see that there's lots of conditions within cells that will favor ATP synthesis even when ATP-ADP ratio is high. All right. This reaction is catalyzed by an enzyme called pyruvate kinase. And pyruvate kinase is a classic example of a, quote-unquote, "irreversible reaction." However, I want to point out that the enzyme pyruvate kinase is actually named for the reverse reaction that's supposedly irreversible. Pyruvate kinase. If you take pyruvate and you act on the kinase as you learn from Professor Yaffe, that phosphorylates something. You phosphorylate pyruvate, you get phosphoenolpyruvate pyruvate kinase. And so remember that absolutely no reaction is reversible. It's only reversed-- what they mean by irreversible when they say pyruvate kinase step in glycolysis is irreversible, is that it is effectively irreversible under the conditions that are compatible with life that exist in cells. Of course, if you have very low ADP and very high ATP, you can come up with a way to net synthesize PEP. You just can't drive the reverse reaction under the ATP-ADP and the PEP pyruvate ratios that physiologically exist in cells. However, this is set up in a way such that you can favor ADP-to-ATP synthesis by converting PEP to pyruvate in a pathway, as we will see happens in glycolysis. All right. Now, the second reaction that I want to-- that allows ATP synthesis in glycolysis is catalyzed by an enzyme called phosphoglycerate kinase. And this carries out the following reaction. OK. So this molecule is 1,3-Bisphosphoglycerate, abbreviated 1,3-BPG. And basically the phosphate is transferred from here to ADP, which gives you this molecule, 3-phosphoglycerate or 3-PG.

~27:44So this reaction, the delta G0 prime for the entire coupled reaction, both steps of the reaction, is minus 4.5 kcals per mole. Tells you about the equilibrium constant. Equilibrium lies to the right, doesn't lie as far to the right as the PEP plus ADP goes to ATP plus pyruvate reaction, but nonetheless, is one where there's lots of conditions that are favorable in cells to maintain a high ATP-ADP ratio, although we will see later that this reaction actually runs quite close to equilibrium in cells, so there's actually conditions where it can go in the opposite direction as well, and we'll get to that later in the course. I want to point out one thing about this, one thing that makes this reaction favorable, as you notice here that this is a phosphate on an acid group, a so-called acid anhydride. And this is a pretty good phosphate donor. And so you want to lose that phosphate, which is one of the reasons why this reaction is favorable for ATP synthesis even at high ATP-ADP ratios. All right. So these are the two reactions in glycolysis that can favor ATP synthesis at a high ATP-ADP ratio just like creatine and creatine phosphate. And so hopefully now it's become apparent to you that if during our pathway of glucose catabolism we can build a pathway that couples what we already know is favorable, glucose oxidation, to generate these intermediates, PEP and 1,3-Bisphosphoglycerate, lacerate we can then build a pathway where those reactions can make synthesis of ATP favorable even though there's a high ATP-ADP ratio in cells, and in fact, maintain that high ATP-ADP ratio in cells. And in essence, this is the logic or the goal, if you will, of glycolysis and why it's important to provide energy to cells. OK. All right. Now, if we're going to make a pathway where glucose oxidation is coupled to making PEP and 1,3Bisphosphoglycerate, it's obvious we need to add phosphate to the system. There's no phosphate molecule on glucose. Now already mentioned earlier-- and I just erased it-- that to trap glucose in the cell, the first step in glycolysis, you add phosphate. But that phosphate donor is ATP. And so ATP being the phosphate donor doesn't help us here. If we're going to use phosphate transfer here, we need to have phosphate added into the system if this is going to work. We can't have ATP as our phosphate donor. You also saw lots of reactions from Professor Yaffe that involved phosphate transfer reactions. But again, ATP-- or GTP is the phosphate donor. And so for this system to work, we need a way to get inorganic phosphate onto a carbohydrate or we can't make things like PEP or 1,3-Bisphosphoglycerate that will allow ATP synthesis. Now we already saw earlier that additional phosphate to glucose is not favored. Remember, delta G0 prime of phosphorylating glucose was plus 3.3 kcals per mole. And it turns out that says equilibrium lies to the wrong side, so we can't net do that without ATP or energy input. And it turns out this is true for other sugars as well. And so you need energy input to add phosphate to the sugars. Plenty of energy input from glucose oxidation, but we need a reaction where phosphate addition is coupled to another reaction that makes it favorable, and we have to do this without making ATP. And again, it illustrates, again, the biological energy is not just about ATP. At a deeper level It's something different. And so to really discuss this and understand it, I need to introduce two concepts that are going to come up again over and over again in this class and across metabolism. The first of these is a very high-level bioenergetic concept, although it's really an energetic concept, and it comes back to what we already talked about.

~31:19So, we know burning wood is favorable. The CN H2O N plus oxygen going to CO2 plus water. That's burning wood, lots of energy released. Drew it up there for a single-glucose molecule and what the energy release the delta G0 prime is for that reaction. Now I want to point out chemically what is going on in this reaction. Chemically what is happening for this to occur is that electrons from the carbon are being moved to the oxygen. You are changing the oxidation state of carbon and of oxygen in order to carry out this reaction. Oxygen is a better electron acceptor than carbon, and so transferring those electrons from the carbon to the oxygen is favorable. That is, in essence, what is going on in this very favorable reaction to burn wood. Let me illustrate that a bit more explicitly here. So the carbon-- most of the carbons in carbohydrates are alcohols. So here, you can call this our carbohydrate, with the exception of the ketone or the aldehyde if it's a ketose or an aldose. All the other carbons in the sugar are alcohols. All the other bonds there. And so if we oxidize that carbon-- that is, if we remove electrons from the carbon, how can we do that? So this here generates this H- hydride ion. This is, in essence, two electrons that we are removing from the carbon. That gives us a ketone. So if we oxidize the alcohol and we remove two electrons, we get the ketone. We could oxidize this ketone further. That will give us the carboxylic acid. If we oxidize it even further, now we get CO2. So, moving in this direction, this is oxidation. Transferring electrons from the carbon to the oxygen to go in this oxidation direction, that is glucose oxidation is what's happening when we're burning wood. It's what happens when we burn glucose. Energy is released. This is largely what catabolism is about in biological systems. Now then reasons, then, that if we're going to move in the opposite direction, so-called reduction, that is going in this direction. Reduction. That's largely energy storage or anabolism in metabolism. We are storing energy that we can burn later. And of course, what's the more extreme version of this? Well, what if we add two more electrons to the carbon going in this direction? Well, what do we end up with? Well, now we end up with the saturated carbon. more reduced than the alcohol is the fully saturated carbon. You will see later that this is what fat is. You saw that in some of the earlier lectures. It's also what gasoline is. What's gasoline? It's fully saturated hydrocarbons that we then burn to CO2 and derive energy. Just like burning wood, burning gasoline all in one step, oxidizing the gasoline gives you energy. There's more energy in gasoline. You can release more energy per weight from gasoline, from fully-saturated hydrocarbons than you can from wood. Same thing happening here. What has more calories in your food? Everyone knows fat has more calories per weight than sugar. It's because the more reduced our carbon is, the more energy it stores, and as we oxidize that carbon, that is what releases the energy. And so energy transduction in biological systems-- and actually, in non-biological systems, as I just illustrated with the wood and gasoline example, but definitely in biological systems, is largely about oxidation and reduction reactions, and you are going to see this coming up over and over again.

~34:34Now, based on what I just said, the real magic, if you will, of glycolysis can be further refined to say that what glycolysis does is it couples the favorable oxidation of carbon with reactions that allow phosphate additions and production of intermediates, like PEP and 1,3-Bisphosphoglycerate, where synthesis of ATP is favored despite the high ATP-ADP ratio in cells. If you think about it, that process then allows you to keep that ATP-ADP ratio in the range where it can then be useful for all the reasons we've been talking about now for two lectures. Allows the cell to couple that ATP-ADP ratio, that-- in ATP hydrolysis to otherwise carry out otherwise unfavorable reactions. OK. So, based on that, arguably the key, or at least a very important reaction in glycolysis, is the oxidation step which happens to add the phosphate and also happens to make 1,3-Bisphosphoglycerate, which we saw earlier can be used to drive ATP synthesis. And so that reaction is the following. So this molecule hopefully looks familiar to you from the carbohydrate lectures. This is D-glyceraldehyde 3phosphate. I will abbreviate it frequently is G-3P for glyceraldehyde 3-phosphate, but it's D-glyceraldehyde 3phosphate. Remember, it's a D, sugar, because the OH group is pointing to the right as we drew it this way. It's also an aldose because it's a aldehyde sugar, and it's phosphorylated on the 3 position-- remember the way we numbered sugars was starting at the carbonyl-- 1, 2, 3. This is carbon-3, phosphorylation on the 3 position. Glyceraldehyde 3-phosphate. OK. So, this step generates 1,3-Bisphosphoglycerate. Well, to do this, there's actually two things that happened here. First we had to add the phosphate to this. It says two phosphates on it. bisphosphoglycerate, this has a single phosphate. So inorganic phosphate has to be added to generate that molecule. By the way, adding that phosphate delta G0 prime is positive plus 1.5 kcals per mole. That means even equilibrium, this reaction would lie to the left, of course, because delta G0 prime is positive. However, remember the next step downstream of this is the one shown over here where we do ATP synthesis. That step is very favorable. So this is an example where metabolism has coupled a very favorable step to a step that's less favorable. That can keep the 1,3-BPG levels low and pull the entire system forward, the metabolic trick we discussed in the last lecture. OK. You'll note the other thing that's happening here, other than adding that phosphate group, is that we've changed this aldehyde to a carboxylic acid. That is an oxidation reaction. Ketone to the acid is an oxidation reaction. I will illustrate over here this so you can see why it's an oxidation reaction. So here's our aldehyde. If we take electrons from the carbon and that generates this hydride ion to electrons, what are we left with? Well, we're left with this intermediate that will never exist with a positive charge on the carbon. Water, which, of course, is abundant in biological systems. And then come-- OK. And it basically illustrates that as I go from the aldehyde to the acid, of course, I make a proton and a hydride ion, but really, I have changed the oxidation state of the carbon from the ketone to the acid state. Two electrons are lost there, is shown as that hydride ion. OK. So, what does that mean? Well, that means that we have to maintain that balance in this reaction as well. So water is going to have to come in and a proton will be produced. But the key thing is we also generate two electrons, this hydride ion that have to go somewhere. Now, finding a place for those two electrons to go is what brings up the second high-level concept that I now need to spend some time discussing.

~38:08And this second high-level concept that I need to introduce is the role of cofactors, and cofactors also is effectively the role of vitamins and what role that these things play in metabolism. Most vitamins are used to support metabolic reactions, and we're going to learn all about what many of the vitamins that you guys are familiar with reading the side of your cereal box actually do throughout the course.

~38:31Now cofactors, as I said, often involve vitamins, are molecules that provide useful chemical groups that facilitate the reaction chemistries that are needed in metabolism. So they provide some useful chemical groups to facilitate some of the chemistries that we need to carry out metabolic reactions. They are, in general, in small non-stoichiometric quantities in order to carry out this function. What do I mean by that? Meaning that you can imagine that the cell will convert lots of glyceraldehyde 3-phosphate to 1,3Bisphosphoglycerate, but the enzyme, you don't need one enzyme for every time you convert that reaction. The same enzyme can do it over and over and over again. Well, it turns out, cofactors are much more like enzymes in that respect. They need to be recycled in a way that they can catalyze the turnover of reaction many, many, many times, and it'll be much clearer what I mean by this when we get into some of the details. Now, the vitamin concept comes from the fact that often these cofactors or parts of the cofactors are not made by animals, not made by humans. If they're not made by us, but we need them to do our metabolism, well now we have to get them from the diet, and this is really the concept of vitamins. These are small things that we have to get in quantities from the diet, and it turns out they're very important to help us carry out the metabolism we do. As an aside, the vitamin industry is a very big business. Lots of unfounded claims out there. I'm not going to get into-- that can get very political. I'm just going to say that just know that more is not always better, and the goal for this class is really to understand what some of these vitamins actually do. OK. Now, the relevant cofactor for the reaction that we're talking about is a cofactor called nicotinamide adenine dinucleotide, which is always abbreviated NAD or NAD+. This stands for nicotinamide adenine dinucleotide. You see why we want to abbreviate that NAD. And basically what nicotinamide adenine dinucleotide is is it's a factor that can solve our problem of where to put these electrons in this oxidation reaction. That is, you can accept two electrons from a reaction like this one here and cycle to another form of the cofactor called NADH. All right. What is nicotinamide adenine dinucleotide? Well first, let's draw what it looks like. OK. Sorry it's squeeze down here a little bit. So this here is adenine. This group up here is nicotinamide. So if you look at this, this is a dinucleotide. And so down here, this, of course, is, from here down, is AMP. That phosphate is now over here. This is a separate nucleotide, where rather than having one of the AGCTU bases that you're used to from RNA and DNA, nicotinamide is the base. And so it's this AMP plus this nicotinamide base is a monophosphate, or a nicotinamide adenine dinucleotide. This, the nicotinamide group, is derived from a vitamin called niacin. And so from the side of your cereal box, this is what niacin does. It allows you to generate the nicotinamide group that's part of this cofactor, nicotinamide adenine dinucleotide. Now, nicotinamide adenine dinucleotide is useful because of what that nicotinamide group can do.

~41:36And so I'm not going to draw out the whole molecule, but I'll show you. So NAD+, which is the oxidized form of nicotinamide adenine dinucleotide, is as I drew it up there. So this here would be attached to the rest of the dinucleotide. So this is the oxidized form. I can have-- here's my hydride ion, which, remember, is two electrons. If I carry out that chemistry to add those two electrons to the nicotinamide group, you now end up with the reduced form of nicotinamide adenine dinucleotide NADH. So the difference between NAD+ and NADH, it's the H, but it's really the two electrons that were added in adding that H. So it's oxidized form and reduced form. So remember, I want to stress this again, at least in biology we can't create or destroy matter. You can do that in nuclear physics, but we can't do that in biology. And so that means that if we do an electron transfer like we did in this reaction where we oxidized the aldehyde in glyceraldehyde 3-phosphate to the acid in 1,3Bisphosphoglycerate, those electrons have to be accounted for. And so if one molecule is oxidized, another one has to be reduced. So redox reactions always-- that is, redox, oxidation-reduction reactions, always have to happen in pairs. And so over there we have the aldehyde in glyceraldehyde 3-phosphate is oxidized to form the acid in 1,3-Bisphosphoglycerate. As part of that reaction, we can couple it such that the nicotinamide group, which is oxidized in NAD, can be reduced to the nicotinamide group that is found in NADH. So that means we can draw this in here, is that having that cofactor coupled to this reaction now allows us to fix the redox problem of this reaction. Now this reaction here is catalyzed by an enzyme called glycerol 3-phosphate dehydrogenase. I'll draw that out in the second. Often abbreviated GAPDH, the loading control in all of your western blots, is the enzyme GAPDH, glyceraldehyde phosphate dehydrogenase. That is the enzyme that carries out that reaction. Now notice, the name fits. It's a dehydrogenase, it's removing a hydrogen, removing two electrons as a hydride ion, but it's really-- what's important here is the electrons moving, not the hydrogen itself. So in that way it's a little bit misleading. And you will see that in general, dehydrogenases are classes of enzymes that catalyze oxidation-reduction reactions, and as a result, often involve NAD/NADH as cofactors because they facilitate those electron transfer reactions. OK. So now we're able to describe how GAPDH works in great detail. And so the chemistry of GAPDH works as follows. And so in the active site of GAPDH, there's a cystine residue. That cystine residue has a sulfur shown here. Also bound near the active site is in NAD molecule to catalyze the reaction shown. That NAD has to be in the oxidized state, so it's NAD+. And so glyceraldehyde 3-phosphate binds in the active site. There's glyceraldehyde 3-phosphate. By the way, I will show you how many reactions in metabolism work chemically. Obviously we don't have time in this class, an overview class to go into all the great chemical details of what's going on and exactly how everything works, but I'll at least try to give you a flavor of some of the chemistry that happens throughout the course. All right. So once you have, then-- OK. So now you have this intermediate bound into the enzyme active site. You can now carry out the oxidation of that carbon. So these are two electrons being removed as a hydride ion. They can be transferred to NAD in the active site. That generates NADH by the exact chemistry that I just showed you. OK.

~45:03That NADH now needs to be exchanged out or the enzyme will not be able to catalyze the next series of reactions, and that NADH will then go somewhere else, and of course, has to be cycled back to NAD, but that doesn't happen in this reaction, per se. OK. Next, this here, you'll notice, is that by the way this chemistry worked, we formed this thioester bond in the active site. This is another thing that you will see over and over again in metabolism. This sulfur in the thioester bond is a good leaving group, and so it's really setting up a situation that will now help with the phosphate addition. So here, I'll draw out inorganic phosphate. And that, of course, will give us the identical enzyme active site that we started with plus 1,3-Bisphosphoglycerate. And that 1,3-Bisphosphoglycerate can now be coupled to ATP synthesis to make 3-phosphoglycerate. And so you can see, in essence, what this reaction has done is that it has coupled oxidation of the aldehyde to the acid, the aldehyde in 3-phosphate to the acid in 1,3-Bisphosphoglycerate, which can then do phosphorylation of ADP to make ATP. So in essence, this is oxidative phosphorylation. Now I realize in high school or in other classes, oxidative phosphorylation means something different to you, and of course, we will discuss the process that is called oxidative phosphorylation in great detail later in this course. But I want to point out that the chemistry that's actually happening here in glycolysis is also oxidative phosphorylation. It's basically how you coupled this favorable carbon oxidation that's occurring at this GAPDH step in a way that allows you to synthesize ATP at a high ATP-ADP ratio, which in the end will allow cells to do otherwise unfavorable things. OK. Now we're ready to discuss these reactions, the GAPDH reaction, the pyruvate kinase reaction, the phosphoglycerate kinase reaction, and how they fit into glycolysis as a metabolic pathway in a way that allows cells to use glucose metabolism-- that is, the breakdown of glucose, the burning of glucose as a way to get energy. And that pathway is, of course, glycolysis or glucose lysis that we mentioned earlier. Glycolysis is a very ancient and ubiquitous pathway. It's used by essentially all life on Earth and probably has been for at least 2 to 3 billion years. Glycolysis, I guess, as a pathway was, quote-unquote, "discovered" mid-19th century by Louis Pasteur, and Louis Pasteur, of course, described life, microorganisms as being responsible for the process of fermentation. That is, conversion of glucose into alcohol, something many college students are interested in, as well as other organic acids. And of course, fermentation to make alcohol or to store other types of organic acids has been used in food preservation for centuries, which is really why Pasteur's discoveries were so impactful at the time. Now even though Pasteur discovered this, he didn't really understand chemically how it worked, and it actually took decades to figure that out. And when it was finally figured out and described as the pathway called glycolysis, this was done by two chemists, two chemists in Germany, Embden and Meyerhof. And basically what they did is they pieced together several enzymatic or chemical activities that have been purified from cell lysates mostly from yeast and used those to build a pathway that described chemically how you could start with glucose and chemically break down that glucose in a way that made sense in this fermentation pathway, and that is why glycolysis is sometimes called the Embden-Meyerhof pathway.

~48:20Now, glycolysis does not completely convert glucose to CO2. Instead, what glycolysis does is it converts glucose-so I will draw here in the pyranose form. It converts glucose by a number of steps into two molecules of the organic acid pyruvate, which, of course, was the product of that pyruvate kinase reaction that we showed earlier. Note that the fermentation that Louis Pasteur described was further metabolism of that pyruvate into something else. In the case of yeast, it was ethanol or ethyl alcohol, or something else. The fermentative product in mammals is this organic acid lactate. And what we will see next time is that this fermentation of pyruvate into one of these other molecules ends up being an alternative to the further oxidation of pyruvate to CO2 which requires oxygen, and the reason oxidizing pyruvate to CO2 requires oxygen is because ultimately we have to regenerate that NADH we made in glycolysis to allow that cofactor to cycle and glycolysis to keep moving. Fermentation gives us an alternative way to do this, which we'll discuss in great detail in the next lecture. But first I want to focus on glycolysis and what we can describe next time as really how we can build a pathway to turn glucose into pyruvate. This glycolysis, this Embden-Meyerhof pathway, and some of the details of that pathway are shown here on this slide. And this is where we'll start off the next lecture, discussing how each of these steps in glycolysis function in a way that allow a pathway to generate phosphoenolpyruvate, 1,3-Bisphosphoglycerate, incorporate this GAPDH reaction, and do so in a way that's overall energetically favorable to allow the cell to couple this oxidation of glucose to keeping ATP-ADP ratio in the right range to support other unfavorable functions in the cell. OK, thanks.

🎬 MIT 7.05 Lec 21 · Glycolysis II & Allosteric/Hormonal Regulation⏱ ~50:00

~00:00MITOCW | 21. Glycolysis II/Regulation

~00:01MATTHEW

~00:02OK. Today we will start our discussion of-- with a discussion of glycolysis as a pathway and how we can use

~00:09VANDER

~00:09favorable oxidation of glucose to enable phosphate addition while at the same time producing useful

~00:15HEIDEN:

~00:15intermediates that enable us to do ATP synthase-- ATP synthesis and maintain a high ATP-ADP ratio in cells. Now I drew up here the entire glycolytic pathway, just like you saw at the end of the last lecture, and I want to go through it here step by step. And so the first step in glycolysis, as we already talked about, is we have glucose. This here is, of course, alpha-D-Glucose in the-- I drew it in the alpha form. If the OH was pointing up, that would be the beta form, just some quick review. I'll drop the alpha and beta and the stereochemistry denotion going forward. This glucose molecule, as we discussed previously, is first trapped in cells by being phosphorylated by the enzyme hexokinase, sometimes abbreviated HK, to give glucose 6-phosphate. Not shown here, this glucose 6phosphate then undergoes an isomerase reaction via the open chain form. Again, we discussed this reaction when we discussed sugars. And so this would be catalyzed by glucose phosphate isomerase, and takes it from the aldose to the ketose, and now you have the fructose-- the ketose fructose 6-phosphate shown here in the furanose form. This fructose 6phosphate is then phosphorylated a second time by an enzyme called phosphofructokinase, typically abbreviated PVK. It's phosphorylated on the 1 position to give fructose 1,6-bisphosphate, shown here. That fructose 1,6-bisphosphate can then be cleaved via the enzyme aldolase. Cleavage takes place in the open chain form which I drew up here in the corner. Basically splitting the molecule in half to form two trioses, one of which is the ketose dihydroxyacetone phosphate, basically the top half of the molecule. The other is the bottom half of the molecule, the aldose glyceraldehyde 3-phosphate. These two trioses can be interconverted, the aldose and the ketose via on isomerase. The enzyme that does that is called trioses phosphate isomerase. And moving forward basically takes the dihydroxyacetone phosphate that comes from the top half and can turn all the carbon from fructose 1,6-bisphosphate into two glyceraldehyde 3phosphates. That glyceraldehyde 3-phosphate, as we discussed last time, is the substrate for the GAPDH reaction, the glyceraldehyde 3-phosphate dehydrogenase, GAPDH. which, as we discussed last time, couples oxidation of this aldehyde to this acid with phosphate addition. That oxidation reaction, the electrons have to go somewhere, that's where the cofactor NAD comes in. Picks up two electrons, generates NADH, and in the process, this enzyme generates this molecule, 1,3-bisphosphoglycerate or 1,3-BPG.

~02:41Remember, that 1,3-BPG molecule was one of the useful intermediates whereby we could undergo favorable synthesis of ATP even at the high ATP-ADP ratio found in cells, and that's because we can transfer the phosphate from this acid group onto ADP. That gives us an ATP and the product 3-phosphoglycerate 3-PG carried out by the enzyme phosphoglycerate kinase. That 3-PG molecule can then undergo what's referred to as a mutase reaction. And so the phosphate is moved from the 3 to the 2 position of glycerate. So 3-phosphoglycerate to 2-phosphoglycerate, carried out by phosphoglycerate mutase, often abbreviated PGAM. This 2-phosphoglycerate can then undergo a dehydration to- by an enzyme enolase to give the molecule phosphoenolpyruvate. . Phosphoenolpyruvate was the second of those useful intermediates that allow ATP synthesis despite the high ATP-ADP ratio in cells, and that reaction, as we already discussed, is carried out by pyruvate kinase and allows you to generate another ATP. Now, as I drew out the glycolytic pathway here, I also listed the delta G0 prime for every single step in the reaction. Remember, this is informative as to the equilibrium for each of these individual reactions. You'll note that for many of the steps, that delta G0 prime is positive. Remember what that means. That means that equilibrium favors the left. That is the reverse direction of the entire pathway. However, if you add all of these up, the delta G0 prime for the entire pathway-- so delta G0 prime for the entire pathway, glucose to pyruvate adds up to negative 6.9 kcals per mole. And so that negative 6.9 means that for the entire pathway, equilibrium favors the right. So glucose to pyruvate, the right direction. And that is why this can work as an overall pathway even though some of the individual steps, equilibrium may favor the opposite direction. Now you'll notice that there's several favorable steps where delta G0 prime is quite negative. Pyruvate kinase, phosphoglycerate kinase, hexokinase, phosphofructokinase. As we talked about before, these are able to pull the less favorable steps upstream forward by keeping concentrations of products such as 1,3-bisphosphoglycerate low, thus allowing this net reaction to move forward. You'll notice I drew three steps here as irreversible-- hexokinase, phosphofructokinase, and pyruvate kinase. Remember, these are all reactions in our-- there's no such thing as an irreversible reaction. There are some conditions where the reverse reaction, of course, can happen. Although remember, this-- when we discussed irreversible in metabolism, what we're referring to is irreversible under physiological conditions-- that is, conditions found in cells. You'll notice that those three steps are three of the four steps that have very negative delta G0 prime. And so you would expect that if we were going to reverse those reactions, you would need some kind of energy input, and you'll see later that these are the control points or also regulated steps in this pathway. And there are also the sites, just to give you a preview, that we're going to have to deal with if we're going to try to synthesize glucose, gluconeogenesis, the reverse of glycolysis.

~05:45Now of course, these delta G0 primes are very informative of what happens at equilibrium, but of course, what actually happens in a cell or in any condition, remember, is dependent on the equilibrium constant, but also on the conditions. Remember, it's delta G, delta G equals delta G0 prime plus RT times the log of the products over the reactants. And so delta G is what really matters for any individual reactions, and what's shown here on this slide is basically a approximate delta G0 change across the glycolytic pathway based on someone's approximation of the conditions found in cells. Now we can see and looking at this that there's really three big drops in delta G across the pathway. Here's the hexokinase reaction, here's the phosphofructokinase reaction, here's the pyruvate kinase reaction. And so it makes sense that these would be, then, the irreversible steps because there is what is actually pulling the pathway forward. It also makes sense that one would exert control at each of these steps, because this is what drives the flux of the pathway forward, but it's also where it's difficult to go back. Once you get here on this flat part of the curve energetically it doesn't matter so much which direction you go along the curve, but to try to climb back up each of these hills becomes difficult. And so we'll revisit this later in the lecture, but this is basically where the control points will end up happening in this pathway. Now, I want to note a couple other features about this pathway, and that is that we had to invest some energy early. You see you cost an ATP at the hexokinase and the phosphofructokinase step and then you harvest that ATP much later at the phosphoglycerate kinase and the pyruvate kinase steps. The textbook typically will split glycolysis into what they refer to as two stages, an investment stage and a harvesting stage, and that basically relates to that fact. And that's because you have a glucose molecule that then you use to generate two trioses. And then each of those trioses can be used to generate a pyruvate molecule. And so it costs 2 ATP to generate those to trioses, but then each triose generated allows you to recapture 2 ATPs. And so what that means, then, is that the net output from glycolysis is 2 ATPs. And so glucose going to two glyceraldehyde 3-phosphates costs you, whereas using those two glyceraldehyde 3-phosphates to generate two pyruvates allows you to recover 4 ATP for a net of plus 2 ATP molecules produced per glucose metabolized. Now if we add up the entire stoichiometry of what's going on here, we can also draw it another way, and that is we can basically have glucose plus 2 ATP molecules plus 2 inorganic phosphates, goes to 2 pyruvates plus 2 ATP, and that's because we're able to incorporate 2 phosphate molecules here at the GAPDH step. Now that's great, but if you're paying attention, you notice that we're missing one other thing in this stoichiometry, and that's that cofactor NAD that was also required to maintain electron balance across the GAPDH step. And so remember that for these pathways to work, we have to balance all things that are going in with all things are going out-- we can't create or destroy matter. And so those electrons have to be dealt with. When we discussed the GAPDH reaction last time, we needed that cofactor NAD in order to act as an acceptor for the electrons to allow that oxidative phosphorylation, if you will, of glyceraldehyde 3-phosphate to 1,3bisphosphoglycerate.

~09:22And so in order for this pathway to work, we need a way to regenerate that NADH back to an NAD in order to make electron balance. In other words, we have to deal with the fact that we've generated-- or that we required 2 NAD+ to run this pathway and generate 2 NADH. And so we need a way to get rid of those electrons from NADH to regenerate that NAD and allow the pathway to be balanced. Now it should be clear that this has to happen stoichiometrically for every single glucose molecule that goes through glycolysis. That is, we have to regenerate 2 NAD plusses for every glucose that we're going to convert into 2 pyruvates. And so for this NADH reoxidation to occur, we need a place to transfer those two electron pairs. And so just like any oxidation and reduction reaction, if we're going to oxidize NADH back to NAD+, something else has to be reduced, and that thing has to be reduced stoichiometrically with every single pyruvate that's produced from glucose and glycolysis. And effectively you will see that this is the role of fermentation. It's an ability for us to solve this redox problem, that we have to dispose of those electrons in the oxidation at the GAPDH step in order to deal with this electron waste of the pathway. Now, the useful way to do this is that we're generating a pyruvate stoichiometrically. And so we can use that stoichiometrically, that product pyruvate by reducing it to form the waste product, and that's effectively what fermentation is. It's turning that pyruvate into ethanol or lactate, which is reducing that pyruvate, picking up the electron waste from the NADH, and allowing you to regenerate the NAD that's required to run the GAPDH step. And so I'll just illustrate this. The simplest fermentation reaction that we can do is the run that's done by animals, and that's to generate lactate. And so to just illustrate this explicitly, here is-- so this here would be the reduced form of NAD, NADH. So this here this-- this is the nicotinamide group. Remember, it's hooked up to an ADP ribose to give nicotinamide adenine dinucleotide in the reduced form. So this can transfer electrons to pyruvate. Here's pyruvate. So as we showed last time, we can take 2 electrons from NADH reduced. That gives us this hydride ion, which can generate lactate. Plus, what we're left with is the oxidized form of the cofactor NAD+. And now this NAD+ can be reduced back to NADH at the GAPDH step, and the electrons from NADH, as it's being reoxidized to NAD, can go and take pyruvate and reduce it to lactate. So NAD gets reoxidized to NAD. Pyruvate now gets-- the ketone gets reduced to the alcohol as you go from pyruvate to lactate. Opposite of what's going on in the GAPDH step. There, the aldehyde is oxidized to the acid, while NAD+ oxidized goes-- is reduced to NADH. Here, NADH is reoxidized to NAD while taking the pyruvate and re-reducing it to lactate. This, by adding here, allows us now to regain balance across the entire pathway. This step is catalyzed by the enzyme lactate dehydrogenase, often abbreviated LDH. And that allows regenerating the NAD+ that's needed for the GAPDH step, which allows us to deal with the stoichiometry problem of the entire pathway. And so what fermentation does for cells is allows electron disposal by taking the product of the pathway pyruvate, reducing it stoichiometrically to form a reduced product-- as shown here, lactate, and that allows you to maintain electron balance across the entire pathway. And so this entire fermentation pathway, glucose to lactate, now allows the generation of the net generation of 2 ATP and run this pathway in cells so cells can maintain a high ATP-ADP ratio by fermenting glucose into lactate.

~13:14Now, if you're paying attention, this also now begins to describe why it is that oxygen is such a useful molecule for metabolism and actually why it's quite key to supporting the bioenergetics of cells. And so recall, oxygen was there when we burned wood and we used that example. And why oxygen is so important for burning wood, why it's important for doing glucose catabolism in our cells is that oxygen is a really great electron acceptor. That is, I can draw out oxygen can take 2 electrons that are generated as NADH waste, if you want to call it that, plus protons, and this generates water. And so an alternative to fermentation in order to allow glucose oxidation is rather than do fermentation, is to transfer those electrons to oxygen to run the pathway another way. In other words, if we write glucose to pyruvate and realize that this is an oxidation reaction, and so therefore, generates electron waste in the form of NADH that has to be recycled to NAD, we can do so by putting those reoxidizing NADH, means something else has to be reduced. This something else could be oxygen as a good electron acceptor to be reduced to water and find an alternative place to put those electrons. Or, if oxygen is not present, now we can do fermentation, turning pyruvate into lactate. It should be clear that if we want to completely oxidize glucose, as in burning wood-- that is, turn it-- all the carbon and glucose into the most oxidized form of carbon, CO2, this also requires places to put electrons. That is, we could generate more NADH. And of course, each of those electrons have to be dealt with. And the final home for those electrons needs to be something that's a good electron acceptor like oxygen, allowing you to continually reoxidize your NADH by reducing oxygen to water. So, in the absence of fermentation, oxygen can be used to maintain electron balance, and of course, much more energy is going to be released if we completely burn glucose than if we only partially oxidize glucose. And this is why oxygen allows more energy to be released, or, as you probably learned in high school, more ATP to be produced from glucose metabolism. Now the details of how all of this works are much more complicated than what I have drawn. Obviously fermentation happens the way I drew it, but all the other details about the role of oxygen and how it fits in to these pathways is more complicated than what I've drawn, and we'll cover this in the upcoming lectures. But the key concept is here, and that is the relationship between oxygen and fermentation is really what's illustrated here. These are two different ways that cells can use to dispose of the electron waste that's produced from carbon oxidation and glucose metabolism. Those electrons have to go somewhere. They can go to the product of glycolysis, say pyruvate to lactate, by lactate dehydrogenase, reduce the pyruvate to lactate, fermentation, or they can go somewhere else. A great electron acceptor like oxygen which allows these pathways to happen while saving the pyruvate to do something else, like be completely oxidized to CO2. Now it should be clear here, then, that one of the things that's special about fermentation is that absolutely no oxygen is required to do this, and this is part of the reason why this pathway is so ancient. Fermentation evolved in the pre-oxygen atmosphere, and that really is why it's such an ancient pathway. Only when oxygen levels rose in the atmosphere due to photosynthesis that enough oxygen was around that then other oxidative pathways could evolve as an alternative to fermentation.

~16:57Now, you'll note that the original description of fermentation by Pasteur was ethanol, not lactate. We'll cover the details of ethanol in a minute, but I just want to point out that making ethanol instead of lactate is simply an alternative product to dispose of pyruvate as a more reduced product. So ethanol is a more reduced product than pyruvate in that ethanol generation is an additional adaptation that allows you to dispose of the electron waste while also generating a molecule that's toxic, and therefore, allow organisms that make ethanol to better kill off their neighbors and their environment and compete for resources. But the overall concept is identical. Ethanol is really an alternative to lactate and fermentation but solves exactly the same problem. I want to talk about ethanol metabolism in more detail, but before we do that first I want to go through and just describe at a very high level some of the chemistry that allows all these reactions in glycolysis to work, because you'll see that the chemistry itself is not all that complicated even though we can draw it and it seems like somewhat overwhelming when drawn out as a whole pathway. Now I want to point out, we've already covered the chemistry for a lot of these steps. First of all, there's two dehydrogenase reactions. There's GAPDH and there's lactate dehydrogenase. Here's the mechanism for lactate dehydrogenase, it's a hydride transfer. The same thing happened at GAPDH with the additional details of adding the phosphate. We covered that previously in an earlier lecture. Note, we also covered before the chemistry of pyruvate kinase and phosphoglycerate kinase. In addition, there's two more kinase reactions that exist here, hexokinase and phosphofructokinase. These are really just straightforward phosphotransfer reactions using ATP to transfer a phosphate. Very straightforward, exactly what Professor Yaffe has already talked to you about, about how one does phosphotransfer for kinase reactions on proteins. Now, two other steps are isomerase reactions, triose phosphatase isomerase and glucose phosphate isomerase. We discussed this chemistry back when we did the sugar lectures. This is just acting on the open chain form of the carbohydrate and allows you to interconvert between the ketose and the aldose as I already drew during that lecture. And so that just leaves a few steps left to discuss that we can cover briefly. The first one that we'll talk about is this enolase reaction, which is simply a dehydration. I'll draw it here just to be explicit as to what's going on. So here is 2-phosphoglycerate. Simply dehydration to remove water, and that gives us phosphoenolpyruvate, PEP. The next step I want to discuss, phosphoglycerate mutase, is an example of a mutase reaction that is, remember, it's moving the phosphate from the 3 to the 2 position of glycerate. This is a class of reactions. It has somewhat of an interesting mechanism. And basically precedes by a phosphorylated enzyme intermediate. In the case of phosphoglycerate mutase, this is a histidine, and so there is a histidine with a nitrogen on it in the active site. And effectively, this nitrogen can pick up a phosphate and be primed via a reaction involving a molecule you've seen already from your discussion of hemoglobin with Professor Yaffe, and that's 2,3-bisphosphoglycerate. And so again, here's-- this is glycerate phosphorylated on the 2 and 3 position. So this is 2,3bisphosphoglycerate. And effectively, this molecule can bind in the active site and pick up a phosphate from either of the 2 or the 3 position. I drew it here is picking it up from the 2 position. That would, of course, generate a 3-phosphoglycerate. But more importantly, it ends up with the enzyme having this phosphorylated intermediate in the active site.

~20:39Once it has that phosphorylated intermediate in the active site, now it's ready to catalyze the mutase reaction. So I'll draw here first, this here would be 3-phosphoglycerate. Color the phosphate so you can see what's going on. This would basically, in the active site of the enzyme, pick up the phosphate from the active site and generate transiently this 2,3-bisphosphoglycerate intermediate, and then it can retransfer the phosphate from the other position back on to the active site of the enzyme so it's ready to carry out another catalytic cycle, and in the process, move the phosphate effectively from the 3 position to the 2 position. So you'll see that the phosphate is not actually being-- the same phosphate is not being moved within the same molecule, it's being ping-ponged back and forth off of this phosphate enzyme intermediate, and basically this is how cells catalyze various mutase reactions to move phosphates between hydroxyl groups. And so you have to prime this once with a separately-generated 2,3-bisphosphoglycerate, but once you have that phosphorylated enzyme in the active site, it can now continually ping-pong that phosphate around to effectively convert 3-phosphoglycerate to 2-phosphoglycerate, or an example of another mutase reaction, move the phosphate between two other hydroxyl groups on the same molecule. OK. Now the final reaction that we haven't talked about is this one, the aldolase reaction, which is a really key step in glycolysis because it splits that carbon-carbon bond to take the hexose-- fructose 1,6-bisphosphate, and split it into two trioses, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, which ultimately allows you to make this pathway work. And so I'll take you quickly through how aldolase works. So aldolase also has this amine in the active site. So here's FBP drawn in the open chain form. So you end up with this intermediate in the active site, which can undergo this chemistry to split this into the-- remove the lower half of the molecule. So you can see here, this generates this lower half of the molecule, glyceraldehyde 3-phosphate, as well as the top half, which can regenerate the active site, as well as the other product, dihydroxyacetone phosphate. And so very briefly, that's effectively the chemistry that allows aldolase to carry out this important reaction in glycolysis, splitting that fructose 1,6-bisphosphate into the aldose, glyceraldehyde 3-phosphate, and the ketose dihydroxyacetone phosphate. OK. So that is effectively the chemistry that allows you to turn glucose into pyruvate, as well as pyruvate into lactate, but what about making ethanol? How does that work? Now ethanol is an alternative fermentative product. And so the reactions to make ethanol is basically this. So here's pyruvate. First thing that happens is we lose this CO2. Without a change in oxidation state at the second carbon at the ketone in pyruvate. And so this ketone stays the same oxidation state, it's now an aldehyde. This is a molecule called acetaldehyde. And now we can pick up those two electrons from NADH and reduce this aldehyde to the alcohol, reoxidizing that NADH back to NAD+ and generating ethyl alcohol or ethanol. So, the fermentative part is turning the acetaldehyde into the ethanol because that allows you to dispose of those two electrons, reduce the aldehyde to an alcohol while reoxidizing the NADH to an NAD+, which is what allows glycolysis to continue so that NAD+ is available for the GAPDH step.

~24:01How this reaction works is-- I just drew it. It's exactly how the lactate dehydrogenase works. However, what we also now need to discuss is how you carry out this reaction, this carboxylation to take this CO2 off of pyruvate and make acetaldehyde. Now decarbonization reactions are really common reactions in metabolism. They end up being important for lots of pathways. Obviously if we're going to completely oxidize glucose into CO2, we have to remove CO2 for a molecule, and here's the first one-- first one that we're going to see for how that happens. Now there's two general mechanisms that metabolism uses for decarboxylation reactions, and I want to introduce the concept here of both of them. Now, the first one here is that these are referred to as decarboxylation reactions that occur in the context of either an alpha keto acid or a beta keto acid. Now, what do I mean by that? Well, pyruvate is an alpha keto acid, and that's because here's a acid group. The ketone is alpha to the carboxylic acid. This would be in contrast to this molecule. So I'll just draw with a generic R group. Where here, the ketone is beta to the carboxylic acid-- alpha, beta. So pyruvate is an alpha keto acid. This here is a generic beta keto acid. Now I want to point out that decarboxylations of bait ketone acids are very favorable, and they're favorable for the following reason. And that's because if I remove this CO2 from a beta keto acid, I'm left with this molecule, which hopefully you'll recognize as an enol. And just like we talked about for the pyruvate kinase reaction, enols much prefer to rearrange into the keto form. And so decarbonization of a beta keto acid generates an enol which will rearrange to the ketone. This becomes very favorable, and so it becomes favorable to decarboxylate a beta keto acid. Now, we can't use that exact same chemistry to decarboxylate an alpha keto acid. However, we can help that process, effectively mimicking the same thing that happens with beta keto acid, by instead introducing a cofactor. So I introduced the concept of cofactors when we talked about NAD and NADH. And remember, cofactors are molecules that provide functional groups that help facilitate the chemistry of various reactions. And so there's a cofactor that facilitates the chemistry that allows decarboxylation of alpha keto acids, and that cofactor is abbreviated TPP+, which stands for the thiamine pyrophosphate. TPP+, which is derived from the vitamin thiamine, sometimes also referred to as the vitamin B1. And so this is what TPP+ looks like. OK. So like many of our vitamins or cofactors, it's a complex molecule. So this is vitamin B1 or thiamine, and the cofactor TPP+ is basically the same molecule with a pyrophosphate group added on the end, TPP+. Now, to see what's going on, we're going to focus here just on this part, this reactive part of the molecule, which is right here, which for simplicity, I will just draw like this. So this here is just this basically part of the molecule here, that's the reactive part of TPP+. And what makes this cofactor useful is that this basically can exist in this stabilized carbanion form. This stabilized carbanion can react with alpha keto acid like pyruvate, shown here. And then this intermediate now becomes favorable for decarboxylation of the CO2 that's alpha to the ketone. And then here, we can regenerate the active TPP+ that we started with plus the other product, acetaldehyde. And then that acetaldehyde can then be reduced, regenerating NAD+ and giving alcohol for fermentation. OK.

~27:38Now incidentally, this is how you produce alcohol. How you metabolize alcohol is effectively reversing that last step and further oxidizing the acetaldehyde. And so this is ethanol. So the way ethanol is metabolized. And so the famous alcohol dehydrogenase enzyme basically does that reverse reaction, generates 2 electrons as a hydride ion. They get transferred to NAD+. That NAD+ gets reduced to NADH as this alcohol in ethanol is oxidized to the aldehyde in acetaldehyde. And this can then be further oxidized, that aldehyde to the acid. So here again, two electrons from the hydride iron can reduce NAD+ to NADH. And that generates, in the process, oxidizes the aldehyde to the acid. In this case, this is acetate. The acid form it would be acetic acid, also known as vinegar. And so effectively, the way you turn alcohol into vinegar is by the microorganisms oxidizing the alcohol they produce into acetic acid or vinegar. Now note, this is-- alcohol metabolism is two oxidation reactions. So we talked about last time, oxidation reactions are generally favorable, therefore energy is released. This is why alcohol has calories and it's impossible to make diet alcohol, because it will-- just no way around this. A couple other things about this, as I mentioned. This enzyme catalyzes. This is the famous alcohol dehydrogenase. This enzyme is an aldehyde dehydrogenase. The issue is is that this aldehyde hydrogenate is a less efficient enzyme than alcohol dehydrogenase, thus it can become rate-limiting for alcohol metabolism. And so if you overwhelm the system by drinking too much, effectively this enzyme can't keep up with this enzyme, you generate excess of the toxic acetaldehyde product. Acetaldehyde product is toxic, and this is effectively what generates hangovers.

~29:21It's also the case that there are individuals with polymorphisms of this aldehyde dehydrogenase. This was, I believe, discussed in Professor Yaffe's lectures on enzyme kinetics that have different catalytic efficiency at this step, and the people who have those polymorphisms can't metabolize alcohol as well. They tend to get flushing and whatnot because they build up this toxic acetaldehyde, and in fact, can be quite dangerous if they drink too much. Finally, this also illustrates effectively why when commercially you want to generate alcohol, like in beer or a wine, you need to keep oxygen levels low. Why is that? Because, well, the microbes will read their ethanol. It's a perfectly good source of calories for them as well because of these reactions. But they generate all this electron waste in the process. Those electrons have to go somewhere. Where do they go? Well, oxygen is a great electron acceptor. And so to metabolize this ethanol, they need to put those electrons somewhere. They need to put them on oxygen. And so oxygen is required to carry out this ethanol metabolism by the microorganisms. And so by keeping oxygen out of your fermentation of alcohol in commercial beer and wine production, it prevents the formation of aldehyde and acetic acid products which would be undesirable in your finished drink product. OK. Now this whole discussion also begins to suggest one big way in which glycolysis itself is regulated, and that's effectively by the availability of oxygen. And so to be explicit about this, I want to draw the relationship between fermentation, glycolysis, and oxygen in terms of maintaining electron balance across these pathways.

~30:58And so glucose is, of course, a major fuel for us. It's a major sugar in our blood. Yeast use a lot of glucose from the grapes in their environment. Plants, of course, make glucose as a storage carbohydrate. It's-- we discussed, starch is a polymer of glucose that uses glucose to survive the night. And effectively what they do is turn that glucose into two pyruvate molecules which can be used to generate net 2 ATP as we discussed. But creates this problem that it also generates NADH that needs to be recycled back to NAD+. In the absence of oxygen, you can ferment that pyruvate into lactate or alcohol or some other product that allows you to regenerate NAD+. Or-- and this is a pathway that requires no oxygen. However, that pyruvate can be further oxidized to CO2, but that's going to generate even more electron waste. That electron waste also has to be dealt with, and that requires oxygen or some other place to put those electrons, oxygen to water being a major driver of further oxidative metabolism. Now the beauty of fermentation as a pathway is that no oxygen is needed, but the trade-off is that it's much less efficient. You only get 2 ATP per mole of glucose that's-- 2 moles of ATP per of glucose that's fermented. You can imagine that a lot more energy is released-- if you can further oxidized glucose, you can generate, therefore, a lot more ATP if you use a complete oxidation of glucose, but this requires oxygen. And so a major way that glycolysis and certainly fermentation is regulated is that low oxygen is what promotes fermentation, whereas high oxygen suppresses fermentation, and that makes sense. It's intuitive. You get a lot more energy. If you completely oxidize your glucose, you can more efficiently get energy from oxidation of available reduced glucose carbon if you do it by oxidative pathways, and so therefore, in the presence of plenty of oxygen, you don't need to do fermentation. Whereas if you outstrip oxygen supply, as, say, might happen if your muscles are exercising, burning more ATP than can be kept up with by oxygen delivery from the blood, now you switch over to a more affirmative metabolism, something like lactic acid builds up that can then be dealt with later. Now, this fact that high oxygen can suppress fermentation is experimentally true in many organisms in many contexts. It's likely because the high ATP-ADP that's produced with oxidative glucose metabolism can directly affect some steps or deplete the NAD that's required to drive glycolysis. Exactly the mechanism that causes this is somewhat debated and likely depends on context and conditions. But it also sets out at least a way for us to begin discussing, how is it at a level of individual enzymes or reactions might something like high ATP-ADP ratio effect flux or flow through a pathway? All right. Well, let's just start by talking about what are the two things that could affect flux or flow through the pathway? Well, remember, thermodynamics, delta G, is what determines whether a pathway happens at all. And because delta G is proportional to things like the ATP-ADP ratio, you can imagine if this ratio gets too high, some of the steps might no longer be favorable to occur. That's certainly one possibility. But the other way that regulation can be done is actually kinetic, and this is because even though enzymes don't determine whether a reaction can happen, at least thermodynamically, they oftentimes do determine the rate of the reaction, and when we're talking about rate and flow through-- flux through pathways, flow through pathways, now rate can become very important.

~34:39And so the kinetics of enzymes, exactly what you talked-- heard about with Professor Yaffe, properties like V max or Km of an enzyme, which, of course, can be affected by allostery, and Professor Yaffe covered in great detail, these also, you can imagine, can affect how a pathway is regulated. And of course, things like ATP, ADP, and AMP as small molecules can be good allosteric affectors of some enzymes. And so let's now talk about, if we're going to regulate steps in a pathway, which steps should actually be regulated. And so shown here is, again, coming back to our delta G change map across glycolysis, as we alluded to earlier, the steps where it's hard to go back is this hexokinase step, this phosphofructokinase step, and this pyruvate kinase step, because these are where there's the big change in delta G that now makes it hard to reverse those steps. And it's exactly those steps that are regulated, because that makes sense. If this delta G is thermodynamically driving flux through the pathway, changing the rate of the enzymes that catalyze those steps is also going to change the rate at which the pathway as a whole can happen. Now as a result, it's these steps, hexokinase, phosphofructokinase, pyruvate kinase, that are often referred to as the rate-limiting steps of glycolysis. Personally, I don't like referring to memorizing things like these are the rate-limiting steps of the pathway, because in reality, what step of any pathway is going to depend on the context. However, often this concept of rate limitation when people refer to pathways really is coming from this energetic argument about which steps have the biggest drop in delta G, and it's important for you to understand that, not just memorize what steps are rate-limiting. Now I also want to point out that these steps actually are in interesting places in glycolysis. So this is basically entry into the pathway, and this is exit from the pathway. And this makes sense, too. You don't want to start committing carbon into a pathway unless you're actually going to need it, and you also need to match output to input. And what you'll see is that many steps in pathways that are regulated, in addition to being the ones with the biggest delta G changes, are also often the entrance and exit from the pathway, which, of course, is the case in glycolysis, but also in most pathways it makes a lot of sense just from logical considerations. All right. Now if we're going to control things that the level of enzymes and rate, how can we control rate of a reaction? That is, how can we control enzyme kinetics? Well, if you remember from Professor Yaffe's lectures, you can increase the rate of a reaction-- that is, catalyzed by an enzyme-- by increasing the Vmax of that enzyme; or, if the substrate concentration is close to Km, by lowering the Km of that enzyme. How can you decrease the rate? Of course, you can do the opposite. You can decrease the Vmax; or, if the substrate concentration is close to Km, you can increase the Km. So how can I change these things? How can I alter Vmax or Km of an individual enzyme? Well, there's a number of ways I can do this. One is is I can make more enzyme. What will making more enzyme do? Well, effectively that will increase the Vmax of an enzyme. Or if I get rid of enzyme, that will decrease the Vmax of an enzyme. I can use a different version of the same enzyme. That is, have an enzyme like pyruvate kinase but have it come in a bunch of different varieties, have different genes encode different pyruvate kinase isoforms such that those isoforms have different Vmax and Km relationships with respect to substrate, and effectively, by using a different version of the enzyme, a so-called isoform of an enzyme, you can now have different properties.

~38:38Now you'll note that the change enzyme kinetics this way basically is new transcription translation. That's fairly long on the timescale of needing to adapt to metabolism, but that can work. But sometimes a lion's attacking you, you need to run away quickly, you may not have time to make more enzyme. You also need direct control of the enzymes, and this is where allosteric regulation of enzyme function comes into play. Allosteric regulation can be a result of signaling. Phosphorylate, acetylate an enzyme, that can change its properties. Or you can have metabolite binding. Bind ATP, bind AMP into some allosteric site that can change Vmax or Km and operate on a short time scale to allow metabolism to adapt in a more acute setting. I want to say that time scales matter for real biology if you want to understand physiology and how it applies to how metabolism and physiology relate to biology that you'll encounter in other contexts. You realize that some responses need to be acute. Those allow you to adapt to conditions quickly, be able to get enough ATP to run away from the lion, et cetera. But you also need adaptations that work on longer time scales, because it's things like this that allow you to enact programs that allow you to adapt long-term to whatever new conditions a cell may face in its environment. All right. Now, before I get into talking exactly how these things actually work to regulate glycolysis, I want to discuss a few details about regulation of pathways in general. And while we talked about these with respect to glycolysis, these points will end up coming up over and over again and really apply to other pathways. Now I may not explicitly point this out, but the same considerations for how glycolysis works, how it's regulated has to be true for all metabolic pathways. And remember, any pathway is just variations on relatively few chemical-- chemistry reactions that are repurposed in a way to build some other thing that's useful to the cell, and you'll see this over and over again. Now, it has to be true for every pathway that it has to be thermodynamically favorable, and that means either that the pathway itself, glucose to pyruvate, is thermodynamically favorable, or it's coupled to some kind of energy input, like ATP hydrolysis that allows the pathway to be favorable. Every pathway is going to be built in a way that intermediates are generated along the way that allow that pathway to accomplish its goals. In the case of glycolysis it was the two steps that allowed us to synthesize and incorporate phosphate despite the high ATP-ADP ratio in cells. And in all cases this has to obey the laws of thermodynamics. That is, we have to obey constant conservation of mass. That means we have to account for all carbon, all electrons, delta G has to be less than 0 across the entire pathway. You'll see that the regulated steps will often be those with the largest thermodynamic considerations, like the big drops across hexokinase, phosphofructokinase, and pyruvate kinase. They'll often be at the entrance and exits to pathway because you want to regulate, you don't want to commit something to a pathway if you're not going to use it, and you want to match input and output. And all of this is going to have regulation that acts on both short and long time scales so it allows it to match physiology. All right. Now with that in mind, now let's discuss how we can regulate glycolysis using both short and long time scales as-- using really short time scales, allosteric regulation of glycolysis in a way that makes it most useful to cells. All right. So the first step is uptake of glucose into the cell, which in animals, like us, is passive, but can still be regulated by glucose transporters, often regulated glute proteins. And it's really these glucose transporters that can often determine whether glucose is actually taken up into cells.

~42:39A famous example of this is FDG PET scanning, which is used to measure glucose uptake into tissues in humans, where basically a hydroxyl group is replaced with a positron-emitting fluorine atom, fluorodeoxyglucose, that is taken up and phosphorylated in cells, and it's this lighting up of this on a positron-emitting scan you can tell that which tissues in the body take up glucose and that's a property of cancer, and so it is often used as a way to detect or stage cancer. Another example of this is insulin. Basically one of the major actions of insulin on your tissues is that when glucose is high, your body makes more insulin. That causes glucose transporters to be put on the surface of your muscle and your fat, which allows that glucose now to get into those cells and effectively allows those cells to dispose of the glucose in the blood and keep glucose in the right physiological range. And so uptake of glucose, at least in animals, is one important step that is really controlled largely by whether or not there is a transporter on the surface. All right. Once that's trapped as-- sorry, once it's in the cell as glucose, hexokinase then traps that molecule as glucose 6-phosphate. And it turns out the glucose 6-phosphate is an inhibitor of hexokinase. Why is that? Well, we will see in the next lecture that glucose 6-phosphate can be turned into glycogen in the case of mammals or starch in the case of plants. And so glucose 6-phosphate, if you're going to store glucose, you can ship it off into glycogen, or you could commit it further down into glycolysis. But if you don't need it for a glycolysis and your stores are full, you'll build up glucose 6-phosphate, you want to inhibit hexokinase to keep things from flowing through the system. The next step that's regulated is phosphofructokinase. So phosphofructokinase, of course, makes FTP. The step downstream of that that's regulated is pyruvate kinase, PEP to pyruvate, and it turns out that FTP will stimulate pyruvate kinase, whereas PEP will inhibit phosphofructokinase. Makes sense. You want to match input to output. If you have a lot of FTP, activate the enzyme to dispose of it. If you're building up PEP, tell you to stop sending molecules down the pipeline. It turns out, PFK is actually the major regulatory step of glycolysis, because this is really what commits that carbon to going through glycolysis as opposed to upstream of that, it could be stored or not. So it's really that step that's a commitment step. And so it's also regulated by some of the outputs of the pathway. And so some of those outputs are ATP downstream of pyruvate, which we'll talk about later in the course. There's some other molecules. Alanine, citrate. It turns out that high levels of citrate will also inhibit PFK. High levels of ATP will inhibit phosphofructokinase. Whereas high levels of AMP will activate phosphofructokinase. Makes sense. A major output of glycolysis is to keep the ATP-ADP ratio high, the energy charge high in the cell. If the energy charge is very high, high ATP, shut off phosphofructokinase, don't need to do more glycolysis. If the energy charge low, high AMP, activate phosphofructokinase. If production of other molecules downstream like citrate are high, come back and stop sending things into the pathway. Turns out, alanine can inhibit pyruvate kinase, another step downstream. Great. This is effectively general regulation, allosteric regulation of how glycolysis works. And what I wrote is was generally true across different tissues, but of course, these regulations can be tweaked to be more or less important to match physiology and function. Now I know many of you are going to take the MCAT. And while I would never want to teach just to take a test, a couple of things that you should remember for that test is phosphofructokinase is a major regulator. Its big allosteric regulators are AMP, as well as ATP and citrate.

~46:38Whereas the other steps, hexokinase, you should know that it's regulated by-- negatively regulated by glucose 6phosphate, and pyruvate kinase is positively regulated by FBP. Now one other point that I want to mention, just because it will come up sometimes on MCAT exams, is that there's an additional detail about how PFK is allosterically regulated, and that's because there's this side reaction where fructose 6-phosphate can be phosphorylated on the 2 rather than the 1 position to make this molecule, fructose 2,6-bisphosphate. This is a reaction that is catalyzed by bifunctional enzyme called PFK-2/FBPase. So basically phosphofructokinase on the 2 position, FBPase, we're moving it from the 2 position which allows you to regulate the levels of fructose 2,6-bisphosphate. It turns out that PFK in glycolysis, which, of course, generates FBP, is positively regulated by fructose 2,6-bisphosphate. And the production of fructose 2,6-bisphosphate is something that can be inhibited by ATP, inhibited by phosphoenolpyruvate, and activated by ATP. So exactly what is shown here is allosteric regulation. Just some of this allosteric regulation is via this side enzyme to make a separate product, fructose 2,6-bisphosphate, and there's control theory considerations for why that might happen. All right. So at this point, we've learned how we can start with glucose, oxidase that glucose via a pathway that now allows us to get the energy that cells need to maintain ATP-ADP ratio in a good physiological range despite the high ATP-ADP ratio in cells. However, it should also be clear that in order for this to work, we need a way to actually get that glucose from somewhere to begin with. You probably learned in high school that plants use photosynthesis as a way to get energy from the sun. Well, the synthesis part of photosynthesis is generating the sugar that those plants can then use to burn and keep ATP-ADP high at night and survive the night until the sun comes up again when they can again use energy from the sun directly to get energy. We as animals evolve the way that we have to eat the plants. We have to get the glucose that the plants make, but of course, we also use-- generate-- have the ability to generate some glucose, and this is best illustrated from muscle physiology. And that is, we alluded to earlier, that if we're running away from the lion, our muscles might outstrip the supply of oxygen in the blood and basically generate a whole lot of lactate. Well, that lactate is perfectly good fuel. Just like the yeast, can oxidize or we can reoxidize ethanol. We can also use lactate as a fuel, but it turns out, a big job of our liver is to take that lactate from the blood, as well as other molecules, and regenerate glucose so that our blood glucose remains in a constant level. This cycling of glucose and lactate across the body between the muscle and the liver is sometimes referred to as the Cori cycle and involves a clear set of reactions to turn that lactate back into glucose, a process called gluconeogenesis. And unfortunately we're out of time today, but next time I will start off the lecture by going through how it is that gluconeogenesis works to turn something like lactate back into glucose so that organisms have the glucose to begin with to run glycolysis. Thank you.

2.4丙酮酸的无氧去路与电子收支平衡：乳酸发酵、TPP 乙醇脱羧与巴斯德效应

一、为什么缺氧时细胞非要把丙酮酸变成“废料”乳酸或乙醇？

在 L21 课堂上，Vander Heiden 教授指着糖酵解的总反应式，提出了一个直击本质的问题：既然糖酵解从葡萄糖到丙酮酸的 10 步反应根本不需要氧气参与，且已经顺利拿到了 2 ATP，为什么在无氧条件下，细胞不能直接把丙酮酸扔掉完事，而非要多此一举再耗费酶去把丙酮酸还原成乳酸或乙醇呢？

答案就在第 6 步（GAPDH）的辅酶循环里！我们在 2.2 节学过，辅酶 NAD⁺ 在细胞质里的总浓度极低（微摩尔级，远少于葡萄糖的消耗量），它就像快递员一样必须循环往复使用。每当 1 分子葡萄糖走完糖酵解，就会在 GAPDH 那一步把 2 NAD⁺ 还原成 2 NADH（教授称之为 “电子废料 Electron waste”）。

• 有氧条件下：世界上最好的电子受体——氧气（O₂） 在线粒体呼吸链终点等着接收这些电子（生成水 H_2O），使胞质 NADH 经穿梭系统源源不断地重新氧化回 NAD⁺，同时把丙酮酸省下来送进线粒体彻底氧化成 CO₂ 赚取海量 ATP。
• 无氧或无线粒体条件下（如剧烈冲刺时的骨骼肌、完全没有线粒体的成熟哺乳动物红细胞、深水缺氧的酵母菌）：没有 O₂ 来接盘电子！如果不能按 1:1 的化学计量比把 NADH 上的电子卸掉，细胞质里有限的 NAD⁺ 库在几秒钟内就会被全部耗尽变成 NADH，第 6 步 GAPDH 因缺乏底物 NAD⁺ 而立即停摆，整个糖酵解和 ATP 生产随之彻底瘫痪！

因此，细胞想出了一个绝妙的“内部消化”方案：既然每生成 2 NADH 刚好同时生成 2 丙酮酸，那就直接拿终产物丙酮酸（或其脱羧产物乙醛）当内源性电子受体，把 NADH 的电子塞回给它！在缺氧条件下，丙酮酸还原为乳酸（或乙醇）本身并不产生任何 ATP，其最核心的生理意义是使 NADH 重新氧化再生为 NAD⁺，以保证第 6 步 3-磷酸甘油醛脱氢酶（GAPDH）持续运转、维持糖酵解不断产生 ATP 2010·判断 2011/2012/2013/2016/2017/2018·单选！这就是生物化学中 发酵（Fermentation） 的严格定义：发酵（fermentation）：在无氧条件下，生物体不经呼吸链和外源电子受体（如 O₂），而是将有机物（如葡萄糖）氧化脱氢释放的电子（NADH）直接交给底物未完全氧化的中间代谢物（如丙酮酸或乙醛），使 NAD⁺ 再生并仅通过底物水平磷酸化获取 ATP 的过程。

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二、无氧去路一：乳酸发酵（Lactic Acid Fermentation）与 LDH 同工酶谱临床诊断

在高等动物组织（尤其是剧烈运动时的骨骼肌、完全无线粒体而只能靠糖酵解生成乳酸供能的成熟哺乳动物红细胞 ⚑华农高频、视网膜、肾髓质）、某些植物器官（如马铃薯块茎、甜菜块根）以及乳酸菌中，丙酮酸在 乳酸脱氢酶（Lactate dehydrogenase, LDH） 催化下，直接接受 NADH 传递的氢负离子（H^-），立体专一地将丙酮酸 C2 酮基还原为 L-乳酸：

丙酮酸 + NADH + H⁺ ⇌(乳酸脱氢酶（LDH）) L-乳酸 + NAD⁺ (Δ G'^° = -25.1 kJ/mol)

把这一步与糖酵解十步反应相加，两边的 2 NAD⁺ 和 2 NADH + 2H⁺ 完美抵消（没有净氧化还原变化！），得到 乳酸发酵总反应式：

葡萄糖 + 2ADP + 2P_i ⟶ 2 L-乳酸 + 2ATP + 2H_2O

3D3D 调节：消化道酶原激活瀑布与同工酶 LDH肠激酶与胰蛋白酶共同激活瀑布、乳酸脱氢酶 LDH1~LDH5 四聚体电泳分离及生理意义↗

关于乳酸脱氢酶（LDH）与乳酸去路，有三个华农考研超高频考点：

1. LDH 四聚体同工酶谱与组织生理分工：同工酶（isozyme / isoenzyme）：指催化相同的化学反应，但酶蛋白的分子结构（氨基酸序列、亚基组成）、理化性质（如电泳迁移率）和动力学参数（如 K_m、V_max）乃至免疫学性质不同的一组酶 ⚑华农高频。如上图所示，哺乳动物的 LDH 是由两种不同基因编码的亚基——H 型（心肌型，Heart）和 M 型（骨骼肌型，Muscle）随机组合而成的四聚体蛋白，共有 5 种同工酶：LDH₁(H₄)、LDH₂(H_3M)、LDH₃(H_2M2)、LDH₄(HM₃)、LDH₅(M₄)。

• 生理逻辑推导（不用死记硬背）：骨骼肌和肝脏富含 M₄（LDH₅）和 M_3H，其对丙酮酸的亲和力高（K_m 小）、不受高浓度丙酮酸抑制，有利于在爆发冲刺缺氧时迅速将丙酮酸还原为乳酸，快速再生 NAD⁺ 供能；而心肌长年进行纯有氧搏动，富含 H₄（LDH₁），其对丙酮酸亲和力低（K_m 大）且受高浓度丙酮酸强烈抑制，但对乳酸亲和力高，从而防止心肌把宝贵的丙酮酸变成乳酸，反而在心肌中催化逆反应将血液运来的乳酸氧化回丙酮酸进入 TCA 循环彻底供能！
• 临床诊断价值：正常人血清中 LDH₂ > LDH₁；当发生急性心肌梗死时，受损心肌释放大量 H₄，导致血清 LDH₁(H₄) 显著飙升并出现 LDH₁ > LDH₂ 的反转；而当发生急性肝炎或骨骼肌损伤时，血清中则是 LDH₅(M₄) 显著升高。

1. 乳酸不是终点垃圾，而是宝贵燃料（Cori 循环）：Vander Heiden 教授在 L21 结尾特别强调，当你逃离狮子后，骨骼肌产生的大量乳酸绝不会被白白尿出体外（乳酸还保留着葡萄糖 93% 以上的化学能！）。注意：LDH 催化的反应是完全可逆的，骨骼肌释放入血的乳酸运往肝脏后，可在肝 LDH 催化下逆向氧化回丙酮酸（同时在肝胞质中生成糖异生第 6 步所需的 NADH），再经糖异生重新合成葡萄糖回补血糖（即 Cori 循环）。
2. 防止乳酸性酸中毒的自保机制：如果剧烈运动时间过长，乳酸大量生成释放 H⁺ 使胞内和血液 pH 下降，升高的 H⁺ 会强烈别构抑制糖酵解限速酶 PFK-1 的活性，从而自动给糖酵解踩刹车，防止机体因乳酸过度堆积而发生致命的酸中毒。

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三、无氧去路二：乙醇发酵（Alcohol Fermentation）、TPP 噻唑环碳负离子脱羧与饮酒代谢

在酵母菌、部分微生物以及缺氧淹水时的植物根系中，细胞采用了另一条更为精巧的两步无氧再生 NAD⁺ 路线——乙醇发酵（人类用于酿酒和发面面包）：

丙酮酸（3C） + H⁺ ──(丙酮酸脱羧酶（辅酶 TPP、Mg²⁺）｜第 1 步：非氧化脱羧（放 CO₂）)──► 乙醛（2C） + CO₂↑

乙醛（2C） + NADH + H⁺ ⇌(乙醇脱氢酶（ADH，含 Zn²⁺）｜第 2 步：还原再生 NAD⁺) 乙醇（2C） + NAD⁺

合并后的 乙醇发酵总反应式：

葡萄糖 + 2ADP + 2P_i ⟶ 2乙醇 + 2CO₂↑ + 2ATP + 2H_2O

1. Vander Heiden 教授的有机化学课：为什么 α-酮酸脱羧必须靠维生素 B₁（TPP）？

在 L21 课堂上，Vander Heiden 教授把 β-酮酸（如草酰乙酸、乙酰乙酸） 和 α-酮酸（如丙酮酸、α-酮戊二酸） 的脱羧难度做了一个绝妙对比：

• β-酮酸为什么极易自发脱羧？ 因为 β-酮酸的羧基脱去 CO₂ 后，留下的那对电子正好可以推向旁边的 β-羰基（C=O），直接形成共振稳定的 烯醇负离子（Enolate） 中间物，随后重排为酮，热力学极其有利！
• α-酮酸（丙酮酸）为什么不能直接脱羧？ 在丙酮酸（CH_3-CO-COO^-）中，羰基就在羧基紧隔壁的 α 位上！如果强行脱去 CO₂，负电荷将直接落在这个羰基碳上形成极度不稳定的酰基碳负离子（CH_3-C̈=O^-），电子根本无处安放（没有电子阱）！
• 焦磷酸硫胺素（TPP）如何把“不可能”变成“可能”？ 为了解决 α-酮酸脱羧缺少电子阱的难题，生物界请出了由 维生素 B₁（硫胺素，Thiamine，缺乏时导致糖代谢受阻、丙酮酸积累引发脚气病 Beriberi / 多发性神经炎）焦磷酸化形成的辅酶——焦磷酸硫胺素（Thiamine pyrophosphate, TPP） 2009/2024·单选 2013·名解！此外，注意：乙醇发酵第一步丙酮酸脱羧酶仅需 TPP（非氧化脱羧，动物体内无此酶），第二步乙醇脱氢酶（ADH）则是含 Zn²⁺ 的金属酶，负责用 NADH 还原乙醛再生 NAD⁺ 2014·单选。

3D3D 分子：焦磷酸硫胺素（TPP）噻唑环 C2 活性位点步骤 1：N⁺ 吸电子诱导 C2—H 解离为亲核碳负离子（叶立德）进攻 α-酮基↗

2. 饮酒脸红、宿醉与为什么造不出“无糖零卡啤酒”？（MIT 7.05 课堂趣味延伸）

讲完酵母如何把丙酮酸变成乙醇来“毒死周围竞争微生物并霸占葡萄汁”后，Vander Heiden 教授顺手在黑板上画出了人类喝下酒（乙醇）之后在肝脏里的逆向拆解路线：

乙醇（Ethanol） + NAD⁺ ──(醇脱氢酶（ADH）)──► 乙醛（Acetaldehyde，剧毒！） + NADH + H⁺

乙醛（Acetaldehyde） + NAD⁺ + H_2O ──(乙醛脱氢酶 2（ALDH2）)──► 乙酸（Acetate / 醋酸） + NADH + H⁺

• 为什么喝酒会脸红和宿醉？ 肝脏里的醇脱氢酶（ADH）催化极快，把乙醇迅速氧化成具有强烈细胞毒性和血管扩张作用的 乙醛；而第二步 线粒体乙醛脱氢酶（ALDH2） 催化效率相对较低，是解酒的限速步。如果你豪饮过量，或者携带东亚人群中高达 30%～50% 频率的 ALDH2 基因单核苷酸多态性突变（Glu487Lys，显性负相失活），乙醛就无法及时转化为无毒的乙酸，在体内大量蓄积，引发毛细血管扩张（“饮酒脸红 Asian flush”）、心动过速、恶心头痛（宿醉 Hangover），并显著增加食管癌风险！
• 为什么永远造不出“零卡路里减肥酒精（Diet Alcohol）”？ 因为乙醇代谢成乙酸（进而活化为乙酰-CoA 进入 TCA 循环）是两步高度放能的碳氧化反应，必然产生大量 NADH 和 ATP（每克酒精释放高达 29 kJ / 7 kcal 热量，仅次于脂肪！），只要喝进肚子被氧化，热力学定律决定了它必定提供高热量！
• 为什么工业酿酒必须严格隔绝空气，一旦漏气酒就会变酸成醋？ 因为酵母和醋酸菌也能通过上述两步氧化反应把乙醇氧化成 乙酸（醋，Vinegar） 来获取能量；但这两步反应每分子乙醇会产生 2 NADH 电子废料，微生物必须有 氧气（O₂） 作为最终电子受体才能跑通乙醇到乙酸的氧化！因此，酿造啤酒和葡萄酒时只要密封隔绝 O₂，微生物就无法进一步氧化乙醇，美酒才不会变成酸醋！

3. 巴斯德效应（Pasteur Effect）：有氧与无氧切换的能量经济学

将有氧氧化（1 分子葡萄糖产 30 或 32 ATP）与无氧发酵（1 分子葡萄糖仅产 2 ATP）放在一起对比，你就立刻明白了路易·巴斯德在研究酵母发酵时发现的经典规律——巴斯德效应（Pasteur effect）：指当微生物（如酵母）或动物组织从无氧条件切换到有氧条件时，由于有氧呼吸产生 ATP 的效率远高于无氧发酵（约 15～16 倍），细胞对葡萄糖的消耗速率显著下降，同时无氧发酵受到抑制、停止积累乙醇或乳酸的现象 ⚑华农高频。其分子机制将在下一节揭晓：有氧氧化产生的大量 ATP 和 柠檬酸（Citrate） 会直接跨过线粒体回到胞质，像两把大锁一样死死关小糖酵解限速酶 PFK-1 的阀门！

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🎧 MIT 7.05 课堂原声英文实录对照（本节关联 1 讲 · 完整逐段实录）点击展开 / 收起 ▾

🎬 MIT 7.05 Lec 21 · Glycolysis II & Allosteric/Hormonal Regulation⏱ ~50:00

~00:00MITOCW | 21. Glycolysis II/Regulation

~00:01MATTHEW

~00:02OK. Today we will start our discussion of-- with a discussion of glycolysis as a pathway and how we can use

~00:09VANDER

~00:09favorable oxidation of glucose to enable phosphate addition while at the same time producing useful

~00:15HEIDEN:

~00:15intermediates that enable us to do ATP synthase-- ATP synthesis and maintain a high ATP-ADP ratio in cells. Now I drew up here the entire glycolytic pathway, just like you saw at the end of the last lecture, and I want to go through it here step by step. And so the first step in glycolysis, as we already talked about, is we have glucose. This here is, of course, alpha-D-Glucose in the-- I drew it in the alpha form. If the OH was pointing up, that would be the beta form, just some quick review. I'll drop the alpha and beta and the stereochemistry denotion going forward. This glucose molecule, as we discussed previously, is first trapped in cells by being phosphorylated by the enzyme hexokinase, sometimes abbreviated HK, to give glucose 6-phosphate. Not shown here, this glucose 6phosphate then undergoes an isomerase reaction via the open chain form. Again, we discussed this reaction when we discussed sugars. And so this would be catalyzed by glucose phosphate isomerase, and takes it from the aldose to the ketose, and now you have the fructose-- the ketose fructose 6-phosphate shown here in the furanose form. This fructose 6phosphate is then phosphorylated a second time by an enzyme called phosphofructokinase, typically abbreviated PVK. It's phosphorylated on the 1 position to give fructose 1,6-bisphosphate, shown here. That fructose 1,6-bisphosphate can then be cleaved via the enzyme aldolase. Cleavage takes place in the open chain form which I drew up here in the corner. Basically splitting the molecule in half to form two trioses, one of which is the ketose dihydroxyacetone phosphate, basically the top half of the molecule. The other is the bottom half of the molecule, the aldose glyceraldehyde 3-phosphate. These two trioses can be interconverted, the aldose and the ketose via on isomerase. The enzyme that does that is called trioses phosphate isomerase. And moving forward basically takes the dihydroxyacetone phosphate that comes from the top half and can turn all the carbon from fructose 1,6-bisphosphate into two glyceraldehyde 3phosphates. That glyceraldehyde 3-phosphate, as we discussed last time, is the substrate for the GAPDH reaction, the glyceraldehyde 3-phosphate dehydrogenase, GAPDH. which, as we discussed last time, couples oxidation of this aldehyde to this acid with phosphate addition. That oxidation reaction, the electrons have to go somewhere, that's where the cofactor NAD comes in. Picks up two electrons, generates NADH, and in the process, this enzyme generates this molecule, 1,3-bisphosphoglycerate or 1,3-BPG.

~02:41Remember, that 1,3-BPG molecule was one of the useful intermediates whereby we could undergo favorable synthesis of ATP even at the high ATP-ADP ratio found in cells, and that's because we can transfer the phosphate from this acid group onto ADP. That gives us an ATP and the product 3-phosphoglycerate 3-PG carried out by the enzyme phosphoglycerate kinase. That 3-PG molecule can then undergo what's referred to as a mutase reaction. And so the phosphate is moved from the 3 to the 2 position of glycerate. So 3-phosphoglycerate to 2-phosphoglycerate, carried out by phosphoglycerate mutase, often abbreviated PGAM. This 2-phosphoglycerate can then undergo a dehydration to- by an enzyme enolase to give the molecule phosphoenolpyruvate. . Phosphoenolpyruvate was the second of those useful intermediates that allow ATP synthesis despite the high ATP-ADP ratio in cells, and that reaction, as we already discussed, is carried out by pyruvate kinase and allows you to generate another ATP. Now, as I drew out the glycolytic pathway here, I also listed the delta G0 prime for every single step in the reaction. Remember, this is informative as to the equilibrium for each of these individual reactions. You'll note that for many of the steps, that delta G0 prime is positive. Remember what that means. That means that equilibrium favors the left. That is the reverse direction of the entire pathway. However, if you add all of these up, the delta G0 prime for the entire pathway-- so delta G0 prime for the entire pathway, glucose to pyruvate adds up to negative 6.9 kcals per mole. And so that negative 6.9 means that for the entire pathway, equilibrium favors the right. So glucose to pyruvate, the right direction. And that is why this can work as an overall pathway even though some of the individual steps, equilibrium may favor the opposite direction. Now you'll notice that there's several favorable steps where delta G0 prime is quite negative. Pyruvate kinase, phosphoglycerate kinase, hexokinase, phosphofructokinase. As we talked about before, these are able to pull the less favorable steps upstream forward by keeping concentrations of products such as 1,3-bisphosphoglycerate low, thus allowing this net reaction to move forward. You'll notice I drew three steps here as irreversible-- hexokinase, phosphofructokinase, and pyruvate kinase. Remember, these are all reactions in our-- there's no such thing as an irreversible reaction. There are some conditions where the reverse reaction, of course, can happen. Although remember, this-- when we discussed irreversible in metabolism, what we're referring to is irreversible under physiological conditions-- that is, conditions found in cells. You'll notice that those three steps are three of the four steps that have very negative delta G0 prime. And so you would expect that if we were going to reverse those reactions, you would need some kind of energy input, and you'll see later that these are the control points or also regulated steps in this pathway. And there are also the sites, just to give you a preview, that we're going to have to deal with if we're going to try to synthesize glucose, gluconeogenesis, the reverse of glycolysis.

~05:45Now of course, these delta G0 primes are very informative of what happens at equilibrium, but of course, what actually happens in a cell or in any condition, remember, is dependent on the equilibrium constant, but also on the conditions. Remember, it's delta G, delta G equals delta G0 prime plus RT times the log of the products over the reactants. And so delta G is what really matters for any individual reactions, and what's shown here on this slide is basically a approximate delta G0 change across the glycolytic pathway based on someone's approximation of the conditions found in cells. Now we can see and looking at this that there's really three big drops in delta G across the pathway. Here's the hexokinase reaction, here's the phosphofructokinase reaction, here's the pyruvate kinase reaction. And so it makes sense that these would be, then, the irreversible steps because there is what is actually pulling the pathway forward. It also makes sense that one would exert control at each of these steps, because this is what drives the flux of the pathway forward, but it's also where it's difficult to go back. Once you get here on this flat part of the curve energetically it doesn't matter so much which direction you go along the curve, but to try to climb back up each of these hills becomes difficult. And so we'll revisit this later in the lecture, but this is basically where the control points will end up happening in this pathway. Now, I want to note a couple other features about this pathway, and that is that we had to invest some energy early. You see you cost an ATP at the hexokinase and the phosphofructokinase step and then you harvest that ATP much later at the phosphoglycerate kinase and the pyruvate kinase steps. The textbook typically will split glycolysis into what they refer to as two stages, an investment stage and a harvesting stage, and that basically relates to that fact. And that's because you have a glucose molecule that then you use to generate two trioses. And then each of those trioses can be used to generate a pyruvate molecule. And so it costs 2 ATP to generate those to trioses, but then each triose generated allows you to recapture 2 ATPs. And so what that means, then, is that the net output from glycolysis is 2 ATPs. And so glucose going to two glyceraldehyde 3-phosphates costs you, whereas using those two glyceraldehyde 3-phosphates to generate two pyruvates allows you to recover 4 ATP for a net of plus 2 ATP molecules produced per glucose metabolized. Now if we add up the entire stoichiometry of what's going on here, we can also draw it another way, and that is we can basically have glucose plus 2 ATP molecules plus 2 inorganic phosphates, goes to 2 pyruvates plus 2 ATP, and that's because we're able to incorporate 2 phosphate molecules here at the GAPDH step. Now that's great, but if you're paying attention, you notice that we're missing one other thing in this stoichiometry, and that's that cofactor NAD that was also required to maintain electron balance across the GAPDH step. And so remember that for these pathways to work, we have to balance all things that are going in with all things are going out-- we can't create or destroy matter. And so those electrons have to be dealt with. When we discussed the GAPDH reaction last time, we needed that cofactor NAD in order to act as an acceptor for the electrons to allow that oxidative phosphorylation, if you will, of glyceraldehyde 3-phosphate to 1,3bisphosphoglycerate.

~09:22And so in order for this pathway to work, we need a way to regenerate that NADH back to an NAD in order to make electron balance. In other words, we have to deal with the fact that we've generated-- or that we required 2 NAD+ to run this pathway and generate 2 NADH. And so we need a way to get rid of those electrons from NADH to regenerate that NAD and allow the pathway to be balanced. Now it should be clear that this has to happen stoichiometrically for every single glucose molecule that goes through glycolysis. That is, we have to regenerate 2 NAD plusses for every glucose that we're going to convert into 2 pyruvates. And so for this NADH reoxidation to occur, we need a place to transfer those two electron pairs. And so just like any oxidation and reduction reaction, if we're going to oxidize NADH back to NAD+, something else has to be reduced, and that thing has to be reduced stoichiometrically with every single pyruvate that's produced from glucose and glycolysis. And effectively you will see that this is the role of fermentation. It's an ability for us to solve this redox problem, that we have to dispose of those electrons in the oxidation at the GAPDH step in order to deal with this electron waste of the pathway. Now, the useful way to do this is that we're generating a pyruvate stoichiometrically. And so we can use that stoichiometrically, that product pyruvate by reducing it to form the waste product, and that's effectively what fermentation is. It's turning that pyruvate into ethanol or lactate, which is reducing that pyruvate, picking up the electron waste from the NADH, and allowing you to regenerate the NAD that's required to run the GAPDH step. And so I'll just illustrate this. The simplest fermentation reaction that we can do is the run that's done by animals, and that's to generate lactate. And so to just illustrate this explicitly, here is-- so this here would be the reduced form of NAD, NADH. So this here this-- this is the nicotinamide group. Remember, it's hooked up to an ADP ribose to give nicotinamide adenine dinucleotide in the reduced form. So this can transfer electrons to pyruvate. Here's pyruvate. So as we showed last time, we can take 2 electrons from NADH reduced. That gives us this hydride ion, which can generate lactate. Plus, what we're left with is the oxidized form of the cofactor NAD+. And now this NAD+ can be reduced back to NADH at the GAPDH step, and the electrons from NADH, as it's being reoxidized to NAD, can go and take pyruvate and reduce it to lactate. So NAD gets reoxidized to NAD. Pyruvate now gets-- the ketone gets reduced to the alcohol as you go from pyruvate to lactate. Opposite of what's going on in the GAPDH step. There, the aldehyde is oxidized to the acid, while NAD+ oxidized goes-- is reduced to NADH. Here, NADH is reoxidized to NAD while taking the pyruvate and re-reducing it to lactate. This, by adding here, allows us now to regain balance across the entire pathway. This step is catalyzed by the enzyme lactate dehydrogenase, often abbreviated LDH. And that allows regenerating the NAD+ that's needed for the GAPDH step, which allows us to deal with the stoichiometry problem of the entire pathway. And so what fermentation does for cells is allows electron disposal by taking the product of the pathway pyruvate, reducing it stoichiometrically to form a reduced product-- as shown here, lactate, and that allows you to maintain electron balance across the entire pathway. And so this entire fermentation pathway, glucose to lactate, now allows the generation of the net generation of 2 ATP and run this pathway in cells so cells can maintain a high ATP-ADP ratio by fermenting glucose into lactate.

~13:14Now, if you're paying attention, this also now begins to describe why it is that oxygen is such a useful molecule for metabolism and actually why it's quite key to supporting the bioenergetics of cells. And so recall, oxygen was there when we burned wood and we used that example. And why oxygen is so important for burning wood, why it's important for doing glucose catabolism in our cells is that oxygen is a really great electron acceptor. That is, I can draw out oxygen can take 2 electrons that are generated as NADH waste, if you want to call it that, plus protons, and this generates water. And so an alternative to fermentation in order to allow glucose oxidation is rather than do fermentation, is to transfer those electrons to oxygen to run the pathway another way. In other words, if we write glucose to pyruvate and realize that this is an oxidation reaction, and so therefore, generates electron waste in the form of NADH that has to be recycled to NAD, we can do so by putting those reoxidizing NADH, means something else has to be reduced. This something else could be oxygen as a good electron acceptor to be reduced to water and find an alternative place to put those electrons. Or, if oxygen is not present, now we can do fermentation, turning pyruvate into lactate. It should be clear that if we want to completely oxidize glucose, as in burning wood-- that is, turn it-- all the carbon and glucose into the most oxidized form of carbon, CO2, this also requires places to put electrons. That is, we could generate more NADH. And of course, each of those electrons have to be dealt with. And the final home for those electrons needs to be something that's a good electron acceptor like oxygen, allowing you to continually reoxidize your NADH by reducing oxygen to water. So, in the absence of fermentation, oxygen can be used to maintain electron balance, and of course, much more energy is going to be released if we completely burn glucose than if we only partially oxidize glucose. And this is why oxygen allows more energy to be released, or, as you probably learned in high school, more ATP to be produced from glucose metabolism. Now the details of how all of this works are much more complicated than what I have drawn. Obviously fermentation happens the way I drew it, but all the other details about the role of oxygen and how it fits in to these pathways is more complicated than what I've drawn, and we'll cover this in the upcoming lectures. But the key concept is here, and that is the relationship between oxygen and fermentation is really what's illustrated here. These are two different ways that cells can use to dispose of the electron waste that's produced from carbon oxidation and glucose metabolism. Those electrons have to go somewhere. They can go to the product of glycolysis, say pyruvate to lactate, by lactate dehydrogenase, reduce the pyruvate to lactate, fermentation, or they can go somewhere else. A great electron acceptor like oxygen which allows these pathways to happen while saving the pyruvate to do something else, like be completely oxidized to CO2. Now it should be clear here, then, that one of the things that's special about fermentation is that absolutely no oxygen is required to do this, and this is part of the reason why this pathway is so ancient. Fermentation evolved in the pre-oxygen atmosphere, and that really is why it's such an ancient pathway. Only when oxygen levels rose in the atmosphere due to photosynthesis that enough oxygen was around that then other oxidative pathways could evolve as an alternative to fermentation.

~16:57Now, you'll note that the original description of fermentation by Pasteur was ethanol, not lactate. We'll cover the details of ethanol in a minute, but I just want to point out that making ethanol instead of lactate is simply an alternative product to dispose of pyruvate as a more reduced product. So ethanol is a more reduced product than pyruvate in that ethanol generation is an additional adaptation that allows you to dispose of the electron waste while also generating a molecule that's toxic, and therefore, allow organisms that make ethanol to better kill off their neighbors and their environment and compete for resources. But the overall concept is identical. Ethanol is really an alternative to lactate and fermentation but solves exactly the same problem. I want to talk about ethanol metabolism in more detail, but before we do that first I want to go through and just describe at a very high level some of the chemistry that allows all these reactions in glycolysis to work, because you'll see that the chemistry itself is not all that complicated even though we can draw it and it seems like somewhat overwhelming when drawn out as a whole pathway. Now I want to point out, we've already covered the chemistry for a lot of these steps. First of all, there's two dehydrogenase reactions. There's GAPDH and there's lactate dehydrogenase. Here's the mechanism for lactate dehydrogenase, it's a hydride transfer. The same thing happened at GAPDH with the additional details of adding the phosphate. We covered that previously in an earlier lecture. Note, we also covered before the chemistry of pyruvate kinase and phosphoglycerate kinase. In addition, there's two more kinase reactions that exist here, hexokinase and phosphofructokinase. These are really just straightforward phosphotransfer reactions using ATP to transfer a phosphate. Very straightforward, exactly what Professor Yaffe has already talked to you about, about how one does phosphotransfer for kinase reactions on proteins. Now, two other steps are isomerase reactions, triose phosphatase isomerase and glucose phosphate isomerase. We discussed this chemistry back when we did the sugar lectures. This is just acting on the open chain form of the carbohydrate and allows you to interconvert between the ketose and the aldose as I already drew during that lecture. And so that just leaves a few steps left to discuss that we can cover briefly. The first one that we'll talk about is this enolase reaction, which is simply a dehydration. I'll draw it here just to be explicit as to what's going on. So here is 2-phosphoglycerate. Simply dehydration to remove water, and that gives us phosphoenolpyruvate, PEP. The next step I want to discuss, phosphoglycerate mutase, is an example of a mutase reaction that is, remember, it's moving the phosphate from the 3 to the 2 position of glycerate. This is a class of reactions. It has somewhat of an interesting mechanism. And basically precedes by a phosphorylated enzyme intermediate. In the case of phosphoglycerate mutase, this is a histidine, and so there is a histidine with a nitrogen on it in the active site. And effectively, this nitrogen can pick up a phosphate and be primed via a reaction involving a molecule you've seen already from your discussion of hemoglobin with Professor Yaffe, and that's 2,3-bisphosphoglycerate. And so again, here's-- this is glycerate phosphorylated on the 2 and 3 position. So this is 2,3bisphosphoglycerate. And effectively, this molecule can bind in the active site and pick up a phosphate from either of the 2 or the 3 position. I drew it here is picking it up from the 2 position. That would, of course, generate a 3-phosphoglycerate. But more importantly, it ends up with the enzyme having this phosphorylated intermediate in the active site.

~20:39Once it has that phosphorylated intermediate in the active site, now it's ready to catalyze the mutase reaction. So I'll draw here first, this here would be 3-phosphoglycerate. Color the phosphate so you can see what's going on. This would basically, in the active site of the enzyme, pick up the phosphate from the active site and generate transiently this 2,3-bisphosphoglycerate intermediate, and then it can retransfer the phosphate from the other position back on to the active site of the enzyme so it's ready to carry out another catalytic cycle, and in the process, move the phosphate effectively from the 3 position to the 2 position. So you'll see that the phosphate is not actually being-- the same phosphate is not being moved within the same molecule, it's being ping-ponged back and forth off of this phosphate enzyme intermediate, and basically this is how cells catalyze various mutase reactions to move phosphates between hydroxyl groups. And so you have to prime this once with a separately-generated 2,3-bisphosphoglycerate, but once you have that phosphorylated enzyme in the active site, it can now continually ping-pong that phosphate around to effectively convert 3-phosphoglycerate to 2-phosphoglycerate, or an example of another mutase reaction, move the phosphate between two other hydroxyl groups on the same molecule. OK. Now the final reaction that we haven't talked about is this one, the aldolase reaction, which is a really key step in glycolysis because it splits that carbon-carbon bond to take the hexose-- fructose 1,6-bisphosphate, and split it into two trioses, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, which ultimately allows you to make this pathway work. And so I'll take you quickly through how aldolase works. So aldolase also has this amine in the active site. So here's FBP drawn in the open chain form. So you end up with this intermediate in the active site, which can undergo this chemistry to split this into the-- remove the lower half of the molecule. So you can see here, this generates this lower half of the molecule, glyceraldehyde 3-phosphate, as well as the top half, which can regenerate the active site, as well as the other product, dihydroxyacetone phosphate. And so very briefly, that's effectively the chemistry that allows aldolase to carry out this important reaction in glycolysis, splitting that fructose 1,6-bisphosphate into the aldose, glyceraldehyde 3-phosphate, and the ketose dihydroxyacetone phosphate. OK. So that is effectively the chemistry that allows you to turn glucose into pyruvate, as well as pyruvate into lactate, but what about making ethanol? How does that work? Now ethanol is an alternative fermentative product. And so the reactions to make ethanol is basically this. So here's pyruvate. First thing that happens is we lose this CO2. Without a change in oxidation state at the second carbon at the ketone in pyruvate. And so this ketone stays the same oxidation state, it's now an aldehyde. This is a molecule called acetaldehyde. And now we can pick up those two electrons from NADH and reduce this aldehyde to the alcohol, reoxidizing that NADH back to NAD+ and generating ethyl alcohol or ethanol. So, the fermentative part is turning the acetaldehyde into the ethanol because that allows you to dispose of those two electrons, reduce the aldehyde to an alcohol while reoxidizing the NADH to an NAD+, which is what allows glycolysis to continue so that NAD+ is available for the GAPDH step.

~24:01How this reaction works is-- I just drew it. It's exactly how the lactate dehydrogenase works. However, what we also now need to discuss is how you carry out this reaction, this carboxylation to take this CO2 off of pyruvate and make acetaldehyde. Now decarbonization reactions are really common reactions in metabolism. They end up being important for lots of pathways. Obviously if we're going to completely oxidize glucose into CO2, we have to remove CO2 for a molecule, and here's the first one-- first one that we're going to see for how that happens. Now there's two general mechanisms that metabolism uses for decarboxylation reactions, and I want to introduce the concept here of both of them. Now, the first one here is that these are referred to as decarboxylation reactions that occur in the context of either an alpha keto acid or a beta keto acid. Now, what do I mean by that? Well, pyruvate is an alpha keto acid, and that's because here's a acid group. The ketone is alpha to the carboxylic acid. This would be in contrast to this molecule. So I'll just draw with a generic R group. Where here, the ketone is beta to the carboxylic acid-- alpha, beta. So pyruvate is an alpha keto acid. This here is a generic beta keto acid. Now I want to point out that decarboxylations of bait ketone acids are very favorable, and they're favorable for the following reason. And that's because if I remove this CO2 from a beta keto acid, I'm left with this molecule, which hopefully you'll recognize as an enol. And just like we talked about for the pyruvate kinase reaction, enols much prefer to rearrange into the keto form. And so decarbonization of a beta keto acid generates an enol which will rearrange to the ketone. This becomes very favorable, and so it becomes favorable to decarboxylate a beta keto acid. Now, we can't use that exact same chemistry to decarboxylate an alpha keto acid. However, we can help that process, effectively mimicking the same thing that happens with beta keto acid, by instead introducing a cofactor. So I introduced the concept of cofactors when we talked about NAD and NADH. And remember, cofactors are molecules that provide functional groups that help facilitate the chemistry of various reactions. And so there's a cofactor that facilitates the chemistry that allows decarboxylation of alpha keto acids, and that cofactor is abbreviated TPP+, which stands for the thiamine pyrophosphate. TPP+, which is derived from the vitamin thiamine, sometimes also referred to as the vitamin B1. And so this is what TPP+ looks like. OK. So like many of our vitamins or cofactors, it's a complex molecule. So this is vitamin B1 or thiamine, and the cofactor TPP+ is basically the same molecule with a pyrophosphate group added on the end, TPP+. Now, to see what's going on, we're going to focus here just on this part, this reactive part of the molecule, which is right here, which for simplicity, I will just draw like this. So this here is just this basically part of the molecule here, that's the reactive part of TPP+. And what makes this cofactor useful is that this basically can exist in this stabilized carbanion form. This stabilized carbanion can react with alpha keto acid like pyruvate, shown here. And then this intermediate now becomes favorable for decarboxylation of the CO2 that's alpha to the ketone. And then here, we can regenerate the active TPP+ that we started with plus the other product, acetaldehyde. And then that acetaldehyde can then be reduced, regenerating NAD+ and giving alcohol for fermentation. OK.

~27:38Now incidentally, this is how you produce alcohol. How you metabolize alcohol is effectively reversing that last step and further oxidizing the acetaldehyde. And so this is ethanol. So the way ethanol is metabolized. And so the famous alcohol dehydrogenase enzyme basically does that reverse reaction, generates 2 electrons as a hydride ion. They get transferred to NAD+. That NAD+ gets reduced to NADH as this alcohol in ethanol is oxidized to the aldehyde in acetaldehyde. And this can then be further oxidized, that aldehyde to the acid. So here again, two electrons from the hydride iron can reduce NAD+ to NADH. And that generates, in the process, oxidizes the aldehyde to the acid. In this case, this is acetate. The acid form it would be acetic acid, also known as vinegar. And so effectively, the way you turn alcohol into vinegar is by the microorganisms oxidizing the alcohol they produce into acetic acid or vinegar. Now note, this is-- alcohol metabolism is two oxidation reactions. So we talked about last time, oxidation reactions are generally favorable, therefore energy is released. This is why alcohol has calories and it's impossible to make diet alcohol, because it will-- just no way around this. A couple other things about this, as I mentioned. This enzyme catalyzes. This is the famous alcohol dehydrogenase. This enzyme is an aldehyde dehydrogenase. The issue is is that this aldehyde hydrogenate is a less efficient enzyme than alcohol dehydrogenase, thus it can become rate-limiting for alcohol metabolism. And so if you overwhelm the system by drinking too much, effectively this enzyme can't keep up with this enzyme, you generate excess of the toxic acetaldehyde product. Acetaldehyde product is toxic, and this is effectively what generates hangovers.

~29:21It's also the case that there are individuals with polymorphisms of this aldehyde dehydrogenase. This was, I believe, discussed in Professor Yaffe's lectures on enzyme kinetics that have different catalytic efficiency at this step, and the people who have those polymorphisms can't metabolize alcohol as well. They tend to get flushing and whatnot because they build up this toxic acetaldehyde, and in fact, can be quite dangerous if they drink too much. Finally, this also illustrates effectively why when commercially you want to generate alcohol, like in beer or a wine, you need to keep oxygen levels low. Why is that? Because, well, the microbes will read their ethanol. It's a perfectly good source of calories for them as well because of these reactions. But they generate all this electron waste in the process. Those electrons have to go somewhere. Where do they go? Well, oxygen is a great electron acceptor. And so to metabolize this ethanol, they need to put those electrons somewhere. They need to put them on oxygen. And so oxygen is required to carry out this ethanol metabolism by the microorganisms. And so by keeping oxygen out of your fermentation of alcohol in commercial beer and wine production, it prevents the formation of aldehyde and acetic acid products which would be undesirable in your finished drink product. OK. Now this whole discussion also begins to suggest one big way in which glycolysis itself is regulated, and that's effectively by the availability of oxygen. And so to be explicit about this, I want to draw the relationship between fermentation, glycolysis, and oxygen in terms of maintaining electron balance across these pathways.

~30:58And so glucose is, of course, a major fuel for us. It's a major sugar in our blood. Yeast use a lot of glucose from the grapes in their environment. Plants, of course, make glucose as a storage carbohydrate. It's-- we discussed, starch is a polymer of glucose that uses glucose to survive the night. And effectively what they do is turn that glucose into two pyruvate molecules which can be used to generate net 2 ATP as we discussed. But creates this problem that it also generates NADH that needs to be recycled back to NAD+. In the absence of oxygen, you can ferment that pyruvate into lactate or alcohol or some other product that allows you to regenerate NAD+. Or-- and this is a pathway that requires no oxygen. However, that pyruvate can be further oxidized to CO2, but that's going to generate even more electron waste. That electron waste also has to be dealt with, and that requires oxygen or some other place to put those electrons, oxygen to water being a major driver of further oxidative metabolism. Now the beauty of fermentation as a pathway is that no oxygen is needed, but the trade-off is that it's much less efficient. You only get 2 ATP per mole of glucose that's-- 2 moles of ATP per of glucose that's fermented. You can imagine that a lot more energy is released-- if you can further oxidized glucose, you can generate, therefore, a lot more ATP if you use a complete oxidation of glucose, but this requires oxygen. And so a major way that glycolysis and certainly fermentation is regulated is that low oxygen is what promotes fermentation, whereas high oxygen suppresses fermentation, and that makes sense. It's intuitive. You get a lot more energy. If you completely oxidize your glucose, you can more efficiently get energy from oxidation of available reduced glucose carbon if you do it by oxidative pathways, and so therefore, in the presence of plenty of oxygen, you don't need to do fermentation. Whereas if you outstrip oxygen supply, as, say, might happen if your muscles are exercising, burning more ATP than can be kept up with by oxygen delivery from the blood, now you switch over to a more affirmative metabolism, something like lactic acid builds up that can then be dealt with later. Now, this fact that high oxygen can suppress fermentation is experimentally true in many organisms in many contexts. It's likely because the high ATP-ADP that's produced with oxidative glucose metabolism can directly affect some steps or deplete the NAD that's required to drive glycolysis. Exactly the mechanism that causes this is somewhat debated and likely depends on context and conditions. But it also sets out at least a way for us to begin discussing, how is it at a level of individual enzymes or reactions might something like high ATP-ADP ratio effect flux or flow through a pathway? All right. Well, let's just start by talking about what are the two things that could affect flux or flow through the pathway? Well, remember, thermodynamics, delta G, is what determines whether a pathway happens at all. And because delta G is proportional to things like the ATP-ADP ratio, you can imagine if this ratio gets too high, some of the steps might no longer be favorable to occur. That's certainly one possibility. But the other way that regulation can be done is actually kinetic, and this is because even though enzymes don't determine whether a reaction can happen, at least thermodynamically, they oftentimes do determine the rate of the reaction, and when we're talking about rate and flow through-- flux through pathways, flow through pathways, now rate can become very important.

~34:39And so the kinetics of enzymes, exactly what you talked-- heard about with Professor Yaffe, properties like V max or Km of an enzyme, which, of course, can be affected by allostery, and Professor Yaffe covered in great detail, these also, you can imagine, can affect how a pathway is regulated. And of course, things like ATP, ADP, and AMP as small molecules can be good allosteric affectors of some enzymes. And so let's now talk about, if we're going to regulate steps in a pathway, which steps should actually be regulated. And so shown here is, again, coming back to our delta G change map across glycolysis, as we alluded to earlier, the steps where it's hard to go back is this hexokinase step, this phosphofructokinase step, and this pyruvate kinase step, because these are where there's the big change in delta G that now makes it hard to reverse those steps. And it's exactly those steps that are regulated, because that makes sense. If this delta G is thermodynamically driving flux through the pathway, changing the rate of the enzymes that catalyze those steps is also going to change the rate at which the pathway as a whole can happen. Now as a result, it's these steps, hexokinase, phosphofructokinase, pyruvate kinase, that are often referred to as the rate-limiting steps of glycolysis. Personally, I don't like referring to memorizing things like these are the rate-limiting steps of the pathway, because in reality, what step of any pathway is going to depend on the context. However, often this concept of rate limitation when people refer to pathways really is coming from this energetic argument about which steps have the biggest drop in delta G, and it's important for you to understand that, not just memorize what steps are rate-limiting. Now I also want to point out that these steps actually are in interesting places in glycolysis. So this is basically entry into the pathway, and this is exit from the pathway. And this makes sense, too. You don't want to start committing carbon into a pathway unless you're actually going to need it, and you also need to match output to input. And what you'll see is that many steps in pathways that are regulated, in addition to being the ones with the biggest delta G changes, are also often the entrance and exit from the pathway, which, of course, is the case in glycolysis, but also in most pathways it makes a lot of sense just from logical considerations. All right. Now if we're going to control things that the level of enzymes and rate, how can we control rate of a reaction? That is, how can we control enzyme kinetics? Well, if you remember from Professor Yaffe's lectures, you can increase the rate of a reaction-- that is, catalyzed by an enzyme-- by increasing the Vmax of that enzyme; or, if the substrate concentration is close to Km, by lowering the Km of that enzyme. How can you decrease the rate? Of course, you can do the opposite. You can decrease the Vmax; or, if the substrate concentration is close to Km, you can increase the Km. So how can I change these things? How can I alter Vmax or Km of an individual enzyme? Well, there's a number of ways I can do this. One is is I can make more enzyme. What will making more enzyme do? Well, effectively that will increase the Vmax of an enzyme. Or if I get rid of enzyme, that will decrease the Vmax of an enzyme. I can use a different version of the same enzyme. That is, have an enzyme like pyruvate kinase but have it come in a bunch of different varieties, have different genes encode different pyruvate kinase isoforms such that those isoforms have different Vmax and Km relationships with respect to substrate, and effectively, by using a different version of the enzyme, a so-called isoform of an enzyme, you can now have different properties.

~38:38Now you'll note that the change enzyme kinetics this way basically is new transcription translation. That's fairly long on the timescale of needing to adapt to metabolism, but that can work. But sometimes a lion's attacking you, you need to run away quickly, you may not have time to make more enzyme. You also need direct control of the enzymes, and this is where allosteric regulation of enzyme function comes into play. Allosteric regulation can be a result of signaling. Phosphorylate, acetylate an enzyme, that can change its properties. Or you can have metabolite binding. Bind ATP, bind AMP into some allosteric site that can change Vmax or Km and operate on a short time scale to allow metabolism to adapt in a more acute setting. I want to say that time scales matter for real biology if you want to understand physiology and how it applies to how metabolism and physiology relate to biology that you'll encounter in other contexts. You realize that some responses need to be acute. Those allow you to adapt to conditions quickly, be able to get enough ATP to run away from the lion, et cetera. But you also need adaptations that work on longer time scales, because it's things like this that allow you to enact programs that allow you to adapt long-term to whatever new conditions a cell may face in its environment. All right. Now, before I get into talking exactly how these things actually work to regulate glycolysis, I want to discuss a few details about regulation of pathways in general. And while we talked about these with respect to glycolysis, these points will end up coming up over and over again and really apply to other pathways. Now I may not explicitly point this out, but the same considerations for how glycolysis works, how it's regulated has to be true for all metabolic pathways. And remember, any pathway is just variations on relatively few chemical-- chemistry reactions that are repurposed in a way to build some other thing that's useful to the cell, and you'll see this over and over again. Now, it has to be true for every pathway that it has to be thermodynamically favorable, and that means either that the pathway itself, glucose to pyruvate, is thermodynamically favorable, or it's coupled to some kind of energy input, like ATP hydrolysis that allows the pathway to be favorable. Every pathway is going to be built in a way that intermediates are generated along the way that allow that pathway to accomplish its goals. In the case of glycolysis it was the two steps that allowed us to synthesize and incorporate phosphate despite the high ATP-ADP ratio in cells. And in all cases this has to obey the laws of thermodynamics. That is, we have to obey constant conservation of mass. That means we have to account for all carbon, all electrons, delta G has to be less than 0 across the entire pathway. You'll see that the regulated steps will often be those with the largest thermodynamic considerations, like the big drops across hexokinase, phosphofructokinase, and pyruvate kinase. They'll often be at the entrance and exits to pathway because you want to regulate, you don't want to commit something to a pathway if you're not going to use it, and you want to match input and output. And all of this is going to have regulation that acts on both short and long time scales so it allows it to match physiology. All right. Now with that in mind, now let's discuss how we can regulate glycolysis using both short and long time scales as-- using really short time scales, allosteric regulation of glycolysis in a way that makes it most useful to cells. All right. So the first step is uptake of glucose into the cell, which in animals, like us, is passive, but can still be regulated by glucose transporters, often regulated glute proteins. And it's really these glucose transporters that can often determine whether glucose is actually taken up into cells.

~42:39A famous example of this is FDG PET scanning, which is used to measure glucose uptake into tissues in humans, where basically a hydroxyl group is replaced with a positron-emitting fluorine atom, fluorodeoxyglucose, that is taken up and phosphorylated in cells, and it's this lighting up of this on a positron-emitting scan you can tell that which tissues in the body take up glucose and that's a property of cancer, and so it is often used as a way to detect or stage cancer. Another example of this is insulin. Basically one of the major actions of insulin on your tissues is that when glucose is high, your body makes more insulin. That causes glucose transporters to be put on the surface of your muscle and your fat, which allows that glucose now to get into those cells and effectively allows those cells to dispose of the glucose in the blood and keep glucose in the right physiological range. And so uptake of glucose, at least in animals, is one important step that is really controlled largely by whether or not there is a transporter on the surface. All right. Once that's trapped as-- sorry, once it's in the cell as glucose, hexokinase then traps that molecule as glucose 6-phosphate. And it turns out the glucose 6-phosphate is an inhibitor of hexokinase. Why is that? Well, we will see in the next lecture that glucose 6-phosphate can be turned into glycogen in the case of mammals or starch in the case of plants. And so glucose 6-phosphate, if you're going to store glucose, you can ship it off into glycogen, or you could commit it further down into glycolysis. But if you don't need it for a glycolysis and your stores are full, you'll build up glucose 6-phosphate, you want to inhibit hexokinase to keep things from flowing through the system. The next step that's regulated is phosphofructokinase. So phosphofructokinase, of course, makes FTP. The step downstream of that that's regulated is pyruvate kinase, PEP to pyruvate, and it turns out that FTP will stimulate pyruvate kinase, whereas PEP will inhibit phosphofructokinase. Makes sense. You want to match input to output. If you have a lot of FTP, activate the enzyme to dispose of it. If you're building up PEP, tell you to stop sending molecules down the pipeline. It turns out, PFK is actually the major regulatory step of glycolysis, because this is really what commits that carbon to going through glycolysis as opposed to upstream of that, it could be stored or not. So it's really that step that's a commitment step. And so it's also regulated by some of the outputs of the pathway. And so some of those outputs are ATP downstream of pyruvate, which we'll talk about later in the course. There's some other molecules. Alanine, citrate. It turns out that high levels of citrate will also inhibit PFK. High levels of ATP will inhibit phosphofructokinase. Whereas high levels of AMP will activate phosphofructokinase. Makes sense. A major output of glycolysis is to keep the ATP-ADP ratio high, the energy charge high in the cell. If the energy charge is very high, high ATP, shut off phosphofructokinase, don't need to do more glycolysis. If the energy charge low, high AMP, activate phosphofructokinase. If production of other molecules downstream like citrate are high, come back and stop sending things into the pathway. Turns out, alanine can inhibit pyruvate kinase, another step downstream. Great. This is effectively general regulation, allosteric regulation of how glycolysis works. And what I wrote is was generally true across different tissues, but of course, these regulations can be tweaked to be more or less important to match physiology and function. Now I know many of you are going to take the MCAT. And while I would never want to teach just to take a test, a couple of things that you should remember for that test is phosphofructokinase is a major regulator. Its big allosteric regulators are AMP, as well as ATP and citrate.

~46:38Whereas the other steps, hexokinase, you should know that it's regulated by-- negatively regulated by glucose 6phosphate, and pyruvate kinase is positively regulated by FBP. Now one other point that I want to mention, just because it will come up sometimes on MCAT exams, is that there's an additional detail about how PFK is allosterically regulated, and that's because there's this side reaction where fructose 6-phosphate can be phosphorylated on the 2 rather than the 1 position to make this molecule, fructose 2,6-bisphosphate. This is a reaction that is catalyzed by bifunctional enzyme called PFK-2/FBPase. So basically phosphofructokinase on the 2 position, FBPase, we're moving it from the 2 position which allows you to regulate the levels of fructose 2,6-bisphosphate. It turns out that PFK in glycolysis, which, of course, generates FBP, is positively regulated by fructose 2,6-bisphosphate. And the production of fructose 2,6-bisphosphate is something that can be inhibited by ATP, inhibited by phosphoenolpyruvate, and activated by ATP. So exactly what is shown here is allosteric regulation. Just some of this allosteric regulation is via this side enzyme to make a separate product, fructose 2,6-bisphosphate, and there's control theory considerations for why that might happen. All right. So at this point, we've learned how we can start with glucose, oxidase that glucose via a pathway that now allows us to get the energy that cells need to maintain ATP-ADP ratio in a good physiological range despite the high ATP-ADP ratio in cells. However, it should also be clear that in order for this to work, we need a way to actually get that glucose from somewhere to begin with. You probably learned in high school that plants use photosynthesis as a way to get energy from the sun. Well, the synthesis part of photosynthesis is generating the sugar that those plants can then use to burn and keep ATP-ADP high at night and survive the night until the sun comes up again when they can again use energy from the sun directly to get energy. We as animals evolve the way that we have to eat the plants. We have to get the glucose that the plants make, but of course, we also use-- generate-- have the ability to generate some glucose, and this is best illustrated from muscle physiology. And that is, we alluded to earlier, that if we're running away from the lion, our muscles might outstrip the supply of oxygen in the blood and basically generate a whole lot of lactate. Well, that lactate is perfectly good fuel. Just like the yeast, can oxidize or we can reoxidize ethanol. We can also use lactate as a fuel, but it turns out, a big job of our liver is to take that lactate from the blood, as well as other molecules, and regenerate glucose so that our blood glucose remains in a constant level. This cycling of glucose and lactate across the body between the muscle and the liver is sometimes referred to as the Cori cycle and involves a clear set of reactions to turn that lactate back into glucose, a process called gluconeogenesis. And unfortunately we're out of time today, but next time I will start off the lecture by going through how it is that gluconeogenesis works to turn something like lactate back into glucose so that organisms have the glucose to begin with to run glycolysis. Thank you.

2.5三大限速酶的多维精密调控：从 HK/GK 组织分工到 PFK-1/F-2,6-BP 双功能酶激素开关

一、代谢调控的总原则：为什么偏偏管住第 1、3、10 这三步？

在 L21 的后半段，Vander Heiden 教授重新调出了糖酵解十步反应的实际自由能（Δ G）走势瀑布图：在全部十步反应中，第 2、4、5、6、7、8、9 这七步反应的胞内实际 Δ G 都非常接近于 0（处于准平衡态，往左往右完全取决于底物与产物的浓度推拉）；唯独 第 1 步（己糖激酶 HK/GK）、第 3 步（磷酸果糖激酶-1 PFK-1）和第 10 步（丙酮酸激酶 PK） 出现了三次断崖式的自由能暴跌（Δ G ≪ 0）！

为什么代谢途径的限速调节位点总是精准选在 Δ G ≪ 0 的不可逆步骤，且恰好分布在途径的入口与出口？① 从热力学上看，只有这些高度放能的“水坝落差处”才是真正拉动整条途径单向流动的驱动力所在，调节它们的酶活性（改变 V_max 或 K_m）才能真正改变整条途径的代谢通量（Flux）；② 从工程设计上看，在第 1 步和第 3 步（入口与承诺步）设卡，可以防止细胞在不需要时盲目把宝贵的葡萄糖投入流水线，而在第 10 步（出口）设卡并与第 3 步联动，则能确保流水线的“产出与投入精准匹配”，防止中间产物堆积或枯竭！

细胞调控这三颗关键酶的手段跨越了两个截然不同的时间尺度：

• 毫秒～秒级（急性短时调控，应对突发狮子袭击）：依靠小分子代谢物（如 ATP、AMP、柠檬酸、F-1,6-BP、F-2,6-BP）对酶的 别构调节（Allosteric regulation），以及激素（胰高血糖素、肾上腺素、胰岛素）触发蛋白激酶/磷酸酶对酶蛋白的 可逆共价修饰（磷酸化/去磷酸化）。
• 小时～天级（慢性长时调控，适应饥饿或高糖饮食）：通过转录因子调控基因转录与翻译（如胰岛素诱导肝细胞合成更多的葡萄糖激酶 GK、PFK-1 和 PK），直接改变细胞内酶蛋白分子的总数量（改变 V_max）或切换不同的同工酶（Isoforms）。

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二、第一道关卡：肌肉己糖激酶（HK I–III）vs 肝脏葡萄糖激酶（GK / HK IV）的精妙分工

人体血液中的正常空腹葡萄糖浓度恒定维持在 4.5～ 5.5 mmol/L 左右。葡萄糖首先通过细胞膜上的葡萄糖转运蛋白（GLUT 家族：脑/红细胞为高亲和力的 GLUT1/GLUT3；骨骼肌和脂肪细胞为受 胰岛素 调节囊泡上膜的 GLUT4；肝细胞和胰岛 β 细胞为高容量、高 K_m 的双向转运体 GLUT2）进入细胞。进胞后的第一步磷酸化，骨骼肌/脑组织与肝脏使用了动力学性质截然不同的两种同工酶——这是华农简答题与单选题最爱考的黄金对比 2008·简答 2012·单选：

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三、第二道总阀门：磷酸果糖激酶-1（PFK-1）的别构网络与 F-2,6-BP 双功能酶共价开关

如果说 HK/GK 是进门的安检员，那么催化第 3 步（F-6-P + ATP → F-1,6-BP + ADP）的 磷酸果糖激酶-1（PFK-1） 就是整条糖酵解无可争议的 最关键限速酶（Pacemaker enzyme） 2009/2015/2024·单选。四聚体别构酶 PFK-1 身上汇集了来自细胞内部能量状态、三羧酸循环负载以及全身内分泌激素的三重指令：

1. 细胞内能量与碳源反馈调控（为什么 ATP 既是底物又是抑制剂？）

• ATP 的双重身份悖论（华农 2012 简答原题）：ATP 明明是 PFK-1 催化反应的必需底物，为什么细胞内高浓度 ATP 反而会强烈抑制 PFK-1？因为 PFK-1 每个亚基上有两个亲和力截然不同的 ATP 结合位点：一个是高亲和力的“催化活性中心（底物位点）”，另一个是低亲和力的“别构调节位点（抑制位点）”。当细胞缺能、ATP 浓度较低时，ATP 优先结合高亲和力的活性中心充当底物，酶处于高活性的 R 态（对底物 F-6-P 呈双曲线动力学）；当细胞能量过剩、ATP 浓度很高时，ATP 结合到低亲和力的别构位点，稳定低活性的 T 态，使酶对另一底物 F-6-P 的亲和力急剧下降（K_0.5 增大，动力学曲线变为典型的 S 形），从而关小糖酵解阀门 2011/2018·判断 2012·简答！
• AMP 与 ADP 的强力激活（解除 ATP 抑制）：当细胞消耗 ATP 时，腺苷酸激酶反应（2ADP ⇌ ATP + AMP）使 AMP 浓度成倍飙升。AMP 和 ADP 是 PFK-1 的别构激活剂，它们能竞争性解除高浓度 ATP 对 PFK-1 的别构抑制，使酶恢复高亲和力的 R 态。
• 柠檬酸（Citrate）的跨区室反馈抑制：当线粒体内三羧酸循环（TCA）运转顺畅、能量和生物合成前体极为充裕时，线粒体基质积累的柠檬酸会通过转运体进入细胞质。细胞质中的柠檬酸是 PFK-1 的重要别构抑制剂（能显著增强 ATP 对 PFK-1 的抑制效应），向糖酵解发出明确信号：“楼下线粒体车间燃料已经满负荷了，请立刻停止往下输送丙酮酸！”（这也是巴斯德效应的核心分子机制之一！）
• H⁺（低 pH）的保护性抑制：无氧酵解产生大量乳酸使胞内 H⁺ 升高时，H⁺ 别构抑制 PFK-1，防止酸中毒。

2. 最强别构激活剂——果糖-2,6-二磷酸（F-2,6-BP）与双功能酶的激素共价开关

在肝脏中，除了看自身能荷高低，更要听从全身血糖指挥官——胰岛素（Insulin，餐后高血糖分泌） 与 胰高血糖素（Glucagon，饥饿低血糖分泌） 的调遣。这两大激素是如何隔空精准操控 PFK-1 的呢？1980 年比利时生化学家 Emile Van Schaftingen 和 Henri-Géry Hers 发现了那个隐藏在幕后的“终极遥控器”——果糖-2,6-二磷酸（Fructose 2,6-bisphosphate, F-2,6-BP）：它是 PFK-1 最强的别构激活剂，同时又是糖异生关键酶果糖-1,6-二磷酸酶-1（FBPase-1）的强效抑制剂 2008·单选 2011/2018·填空！

请千万注意区分这对外表极像的“孪生兄弟”：

• 果糖-1,6-二磷酸（F-1,6-BP）是 PFK-1 的催化产物和醛缩酶的底物，走糖酵解主干道（它绝不反馈抑制 PFK-1，反而前馈激活下游的丙酮酸激酶 PK！） 2016·判断；
• 果糖-2,6-二磷酸（F-2,6-BP）不是糖酵解的中间产物，而是由侧支上的“双功能酶”专门合成的纯调节信号分子！哪怕在微摩尔（μmol/L）极低浓度下，F-2,6-BP 也能大幅提高 PFK-1 对底物 F-6-P 的亲和力（使 S 形曲线变回高斜率双曲线），并彻底解除高浓度 ATP 和柠檬酸对 PFK-1 的抑制！

那么，细胞内 F-2,6-BP 的浓度又是谁来控制的呢？答案是一条极其罕见的、把两个功能完全相反的酶活性中心串在同一条多肽链上的 双功能酶（Bifunctional enzyme / Tandem enzyme）：磷酸果糖激酶-2 / 果糖-2,6-二磷酸酶-2（PFK-2 / FBPase-2）：

• 该多肽链的 N 端结构域是激酶 PFK-2（催化 F-6-P + ATP → F-2,6-BP + ADP，负责合成 F-2,6-BP）；
• 该多肽链的 C 端结构域是磷酸酶 FBPase-2（催化 F-2,6-BP + H_2O → F-6-P + P_i，负责水解销毁 F-2,6-BP）。
• 一个丝氨酸残基（Ser32）磷酸化决定生死的“跷跷板开关”：

1. 饥饿 / 低血糖状态（胰高血糖素主导）：血糖下降 → 胰岛 α 细胞分泌 胰高血糖素（Glucagon） → 肝细胞膜受体激活腺苷酸环化酶 → 胞内 cAMP↑ → 激活 蛋白激酶 A（PKA） → PKA 催化肝双功能酶（PFK-2/FBPase-2）N 端的 Ser32 发生磷酸化 → 构象翻转使 PFK-2 激酶活性失活、FBPase-2 磷酸酶活性激活 → F-2,6-BP 被迅速水解而浓度暴跌（[F-2,6-BP]↓） → PFK-1 失去激活剂而关闭（肝糖酵解踩刹车），同时 FBPase-1 解除抑制而开启（肝糖异生全力开动，向血液输出葡萄糖救济大脑）！
2. 饱餐 / 高血糖状态（胰岛素与戊糖磷酸途径协同主导）：血糖升高 → 胰岛 β 细胞分泌 胰岛素（Insulin）（同时肝内高葡萄糖走戊糖磷酸途径产生大量 5-磷酸木酮糖 Xu-5-P） → 共同激活 磷蛋白磷酸酶 2A（PP2A） → 催化双功能酶 去磷酸化 → PFK-2 激酶激活、FBPase-2 磷酸酶失活（且高浓度底物 F-6-P 本身也别构激活 PFK-2 并抑制 FBPase-2） → F-2,6-BP 浓度飙升（[F-2,6-BP]↑） → 强力激活 PFK-1（肝糖酵解全速开启，将多余血糖转化为丙酮酸和乙酰-CoA 合成脂肪储存），同时锁死糖异生！

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四、第三道出口阀：丙酮酸激酶（PK）的前馈激活与肝 L-PK 的共价制动

在糖酵解的最后一步（第 10 步，PEP + ADP → 丙酮酸 + ATP），把守出口的 丙酮酸激酶（Pyruvate kinase, PK） 同样受到别构与激素共价修饰的双重严密管控：

1. 所有组织 PK 共有的别构调节（前馈激活与产物反馈抑制）：

• 果糖-1,6-二磷酸（F-1,6-BP）的前馈激活（Feed-forward activation）：这是代谢网络中最经典的“首尾呼应”设计！当第 3 步 PFK-1 开闸放水生成大量 F-1,6-BP 时，上游产物 F-1,6-BP 会直接跨越 7 步反应，作为强效别构激活剂提前唤醒第 10 步的丙酮酸激酶（PK）（同时底物 PEP 也有正协同激活作用）。Vander Heiden 教授将其比作工厂流水线调度：一旦车头（PFK-1）开始进料，立刻打电话通知车尾（PK）开足马力卸货，从而保证中游的磷酸丙糖和 PEP 不会发生交通堵塞！（反过来，如果 PEP 堆积，也会逆向抑制 PFK-1）。
• 负反馈别构抑制剂：当细胞能量充沛或下游合成原料饱和时，高浓度的 ATP、乙酰-CoA、长链脂肪酸以及丙氨酸（Alanine，由丙酮酸一步转氨生成，代表氨基酸构建模块充足）均会别构抑制丙酮酸激酶（PK），关小出口阀门。

1. 肝脏特异的激素共价修饰（肝 L 型 vs 肌肉 M 型）：

• 脊椎动物体内有多种 PK 同工酶，其中肝脏表达 L 型（L-PK），肌肉和脑表达 M 型（M-PK）。
• 为什么只有肝 L-PK 带有 PKA 磷酸化位点？ 设想你在饥饿低血糖时，肝脏正在辛辛苦苦消耗 GTP 把草酰乙酸变成 PEP 以进行糖异生；如果此时肝脏里的丙酮酸激酶（PK）还开着，刚造出来的 PEP 就会立刻被 PK 一脚踹回丙酮酸，形成白白浪费能量的“无效循环（Futile cycle）”！因此，在低血糖时，胰高血糖素经 cAMP 激活 PKA，直接催化肝脏 L 型丙酮酸激酶（L-PK）发生磷酸化而失活（胰岛素则促使其去磷酸化恢复活性）；而骨骼肌 M 型丙酮酸激酶（M-PK）无此磷酸化位点，不受胰高血糖素抑制——这样既保证了肝脏在低血糖时绝不偷吃正在异生的 PEP（把葡萄糖省给大脑），又不影响骨骼肌随时用糖酵解供能！

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🎧 MIT 7.05 课堂原声英文实录对照（本节关联 1 讲 · 完整逐段实录）点击展开 / 收起 ▾

🎬 MIT 7.05 Lec 21 · Glycolysis II & Allosteric/Hormonal Regulation⏱ ~50:00

~00:00MITOCW | 21. Glycolysis II/Regulation

~00:01MATTHEW

~00:02OK. Today we will start our discussion of-- with a discussion of glycolysis as a pathway and how we can use

~00:09VANDER

~00:09favorable oxidation of glucose to enable phosphate addition while at the same time producing useful

~00:15HEIDEN:

~00:15intermediates that enable us to do ATP synthase-- ATP synthesis and maintain a high ATP-ADP ratio in cells. Now I drew up here the entire glycolytic pathway, just like you saw at the end of the last lecture, and I want to go through it here step by step. And so the first step in glycolysis, as we already talked about, is we have glucose. This here is, of course, alpha-D-Glucose in the-- I drew it in the alpha form. If the OH was pointing up, that would be the beta form, just some quick review. I'll drop the alpha and beta and the stereochemistry denotion going forward. This glucose molecule, as we discussed previously, is first trapped in cells by being phosphorylated by the enzyme hexokinase, sometimes abbreviated HK, to give glucose 6-phosphate. Not shown here, this glucose 6phosphate then undergoes an isomerase reaction via the open chain form. Again, we discussed this reaction when we discussed sugars. And so this would be catalyzed by glucose phosphate isomerase, and takes it from the aldose to the ketose, and now you have the fructose-- the ketose fructose 6-phosphate shown here in the furanose form. This fructose 6phosphate is then phosphorylated a second time by an enzyme called phosphofructokinase, typically abbreviated PVK. It's phosphorylated on the 1 position to give fructose 1,6-bisphosphate, shown here. That fructose 1,6-bisphosphate can then be cleaved via the enzyme aldolase. Cleavage takes place in the open chain form which I drew up here in the corner. Basically splitting the molecule in half to form two trioses, one of which is the ketose dihydroxyacetone phosphate, basically the top half of the molecule. The other is the bottom half of the molecule, the aldose glyceraldehyde 3-phosphate. These two trioses can be interconverted, the aldose and the ketose via on isomerase. The enzyme that does that is called trioses phosphate isomerase. And moving forward basically takes the dihydroxyacetone phosphate that comes from the top half and can turn all the carbon from fructose 1,6-bisphosphate into two glyceraldehyde 3phosphates. That glyceraldehyde 3-phosphate, as we discussed last time, is the substrate for the GAPDH reaction, the glyceraldehyde 3-phosphate dehydrogenase, GAPDH. which, as we discussed last time, couples oxidation of this aldehyde to this acid with phosphate addition. That oxidation reaction, the electrons have to go somewhere, that's where the cofactor NAD comes in. Picks up two electrons, generates NADH, and in the process, this enzyme generates this molecule, 1,3-bisphosphoglycerate or 1,3-BPG.

~02:41Remember, that 1,3-BPG molecule was one of the useful intermediates whereby we could undergo favorable synthesis of ATP even at the high ATP-ADP ratio found in cells, and that's because we can transfer the phosphate from this acid group onto ADP. That gives us an ATP and the product 3-phosphoglycerate 3-PG carried out by the enzyme phosphoglycerate kinase. That 3-PG molecule can then undergo what's referred to as a mutase reaction. And so the phosphate is moved from the 3 to the 2 position of glycerate. So 3-phosphoglycerate to 2-phosphoglycerate, carried out by phosphoglycerate mutase, often abbreviated PGAM. This 2-phosphoglycerate can then undergo a dehydration to- by an enzyme enolase to give the molecule phosphoenolpyruvate. . Phosphoenolpyruvate was the second of those useful intermediates that allow ATP synthesis despite the high ATP-ADP ratio in cells, and that reaction, as we already discussed, is carried out by pyruvate kinase and allows you to generate another ATP. Now, as I drew out the glycolytic pathway here, I also listed the delta G0 prime for every single step in the reaction. Remember, this is informative as to the equilibrium for each of these individual reactions. You'll note that for many of the steps, that delta G0 prime is positive. Remember what that means. That means that equilibrium favors the left. That is the reverse direction of the entire pathway. However, if you add all of these up, the delta G0 prime for the entire pathway-- so delta G0 prime for the entire pathway, glucose to pyruvate adds up to negative 6.9 kcals per mole. And so that negative 6.9 means that for the entire pathway, equilibrium favors the right. So glucose to pyruvate, the right direction. And that is why this can work as an overall pathway even though some of the individual steps, equilibrium may favor the opposite direction. Now you'll notice that there's several favorable steps where delta G0 prime is quite negative. Pyruvate kinase, phosphoglycerate kinase, hexokinase, phosphofructokinase. As we talked about before, these are able to pull the less favorable steps upstream forward by keeping concentrations of products such as 1,3-bisphosphoglycerate low, thus allowing this net reaction to move forward. You'll notice I drew three steps here as irreversible-- hexokinase, phosphofructokinase, and pyruvate kinase. Remember, these are all reactions in our-- there's no such thing as an irreversible reaction. There are some conditions where the reverse reaction, of course, can happen. Although remember, this-- when we discussed irreversible in metabolism, what we're referring to is irreversible under physiological conditions-- that is, conditions found in cells. You'll notice that those three steps are three of the four steps that have very negative delta G0 prime. And so you would expect that if we were going to reverse those reactions, you would need some kind of energy input, and you'll see later that these are the control points or also regulated steps in this pathway. And there are also the sites, just to give you a preview, that we're going to have to deal with if we're going to try to synthesize glucose, gluconeogenesis, the reverse of glycolysis.

~05:45Now of course, these delta G0 primes are very informative of what happens at equilibrium, but of course, what actually happens in a cell or in any condition, remember, is dependent on the equilibrium constant, but also on the conditions. Remember, it's delta G, delta G equals delta G0 prime plus RT times the log of the products over the reactants. And so delta G is what really matters for any individual reactions, and what's shown here on this slide is basically a approximate delta G0 change across the glycolytic pathway based on someone's approximation of the conditions found in cells. Now we can see and looking at this that there's really three big drops in delta G across the pathway. Here's the hexokinase reaction, here's the phosphofructokinase reaction, here's the pyruvate kinase reaction. And so it makes sense that these would be, then, the irreversible steps because there is what is actually pulling the pathway forward. It also makes sense that one would exert control at each of these steps, because this is what drives the flux of the pathway forward, but it's also where it's difficult to go back. Once you get here on this flat part of the curve energetically it doesn't matter so much which direction you go along the curve, but to try to climb back up each of these hills becomes difficult. And so we'll revisit this later in the lecture, but this is basically where the control points will end up happening in this pathway. Now, I want to note a couple other features about this pathway, and that is that we had to invest some energy early. You see you cost an ATP at the hexokinase and the phosphofructokinase step and then you harvest that ATP much later at the phosphoglycerate kinase and the pyruvate kinase steps. The textbook typically will split glycolysis into what they refer to as two stages, an investment stage and a harvesting stage, and that basically relates to that fact. And that's because you have a glucose molecule that then you use to generate two trioses. And then each of those trioses can be used to generate a pyruvate molecule. And so it costs 2 ATP to generate those to trioses, but then each triose generated allows you to recapture 2 ATPs. And so what that means, then, is that the net output from glycolysis is 2 ATPs. And so glucose going to two glyceraldehyde 3-phosphates costs you, whereas using those two glyceraldehyde 3-phosphates to generate two pyruvates allows you to recover 4 ATP for a net of plus 2 ATP molecules produced per glucose metabolized. Now if we add up the entire stoichiometry of what's going on here, we can also draw it another way, and that is we can basically have glucose plus 2 ATP molecules plus 2 inorganic phosphates, goes to 2 pyruvates plus 2 ATP, and that's because we're able to incorporate 2 phosphate molecules here at the GAPDH step. Now that's great, but if you're paying attention, you notice that we're missing one other thing in this stoichiometry, and that's that cofactor NAD that was also required to maintain electron balance across the GAPDH step. And so remember that for these pathways to work, we have to balance all things that are going in with all things are going out-- we can't create or destroy matter. And so those electrons have to be dealt with. When we discussed the GAPDH reaction last time, we needed that cofactor NAD in order to act as an acceptor for the electrons to allow that oxidative phosphorylation, if you will, of glyceraldehyde 3-phosphate to 1,3bisphosphoglycerate.

~09:22And so in order for this pathway to work, we need a way to regenerate that NADH back to an NAD in order to make electron balance. In other words, we have to deal with the fact that we've generated-- or that we required 2 NAD+ to run this pathway and generate 2 NADH. And so we need a way to get rid of those electrons from NADH to regenerate that NAD and allow the pathway to be balanced. Now it should be clear that this has to happen stoichiometrically for every single glucose molecule that goes through glycolysis. That is, we have to regenerate 2 NAD plusses for every glucose that we're going to convert into 2 pyruvates. And so for this NADH reoxidation to occur, we need a place to transfer those two electron pairs. And so just like any oxidation and reduction reaction, if we're going to oxidize NADH back to NAD+, something else has to be reduced, and that thing has to be reduced stoichiometrically with every single pyruvate that's produced from glucose and glycolysis. And effectively you will see that this is the role of fermentation. It's an ability for us to solve this redox problem, that we have to dispose of those electrons in the oxidation at the GAPDH step in order to deal with this electron waste of the pathway. Now, the useful way to do this is that we're generating a pyruvate stoichiometrically. And so we can use that stoichiometrically, that product pyruvate by reducing it to form the waste product, and that's effectively what fermentation is. It's turning that pyruvate into ethanol or lactate, which is reducing that pyruvate, picking up the electron waste from the NADH, and allowing you to regenerate the NAD that's required to run the GAPDH step. And so I'll just illustrate this. The simplest fermentation reaction that we can do is the run that's done by animals, and that's to generate lactate. And so to just illustrate this explicitly, here is-- so this here would be the reduced form of NAD, NADH. So this here this-- this is the nicotinamide group. Remember, it's hooked up to an ADP ribose to give nicotinamide adenine dinucleotide in the reduced form. So this can transfer electrons to pyruvate. Here's pyruvate. So as we showed last time, we can take 2 electrons from NADH reduced. That gives us this hydride ion, which can generate lactate. Plus, what we're left with is the oxidized form of the cofactor NAD+. And now this NAD+ can be reduced back to NADH at the GAPDH step, and the electrons from NADH, as it's being reoxidized to NAD, can go and take pyruvate and reduce it to lactate. So NAD gets reoxidized to NAD. Pyruvate now gets-- the ketone gets reduced to the alcohol as you go from pyruvate to lactate. Opposite of what's going on in the GAPDH step. There, the aldehyde is oxidized to the acid, while NAD+ oxidized goes-- is reduced to NADH. Here, NADH is reoxidized to NAD while taking the pyruvate and re-reducing it to lactate. This, by adding here, allows us now to regain balance across the entire pathway. This step is catalyzed by the enzyme lactate dehydrogenase, often abbreviated LDH. And that allows regenerating the NAD+ that's needed for the GAPDH step, which allows us to deal with the stoichiometry problem of the entire pathway. And so what fermentation does for cells is allows electron disposal by taking the product of the pathway pyruvate, reducing it stoichiometrically to form a reduced product-- as shown here, lactate, and that allows you to maintain electron balance across the entire pathway. And so this entire fermentation pathway, glucose to lactate, now allows the generation of the net generation of 2 ATP and run this pathway in cells so cells can maintain a high ATP-ADP ratio by fermenting glucose into lactate.

~13:14Now, if you're paying attention, this also now begins to describe why it is that oxygen is such a useful molecule for metabolism and actually why it's quite key to supporting the bioenergetics of cells. And so recall, oxygen was there when we burned wood and we used that example. And why oxygen is so important for burning wood, why it's important for doing glucose catabolism in our cells is that oxygen is a really great electron acceptor. That is, I can draw out oxygen can take 2 electrons that are generated as NADH waste, if you want to call it that, plus protons, and this generates water. And so an alternative to fermentation in order to allow glucose oxidation is rather than do fermentation, is to transfer those electrons to oxygen to run the pathway another way. In other words, if we write glucose to pyruvate and realize that this is an oxidation reaction, and so therefore, generates electron waste in the form of NADH that has to be recycled to NAD, we can do so by putting those reoxidizing NADH, means something else has to be reduced. This something else could be oxygen as a good electron acceptor to be reduced to water and find an alternative place to put those electrons. Or, if oxygen is not present, now we can do fermentation, turning pyruvate into lactate. It should be clear that if we want to completely oxidize glucose, as in burning wood-- that is, turn it-- all the carbon and glucose into the most oxidized form of carbon, CO2, this also requires places to put electrons. That is, we could generate more NADH. And of course, each of those electrons have to be dealt with. And the final home for those electrons needs to be something that's a good electron acceptor like oxygen, allowing you to continually reoxidize your NADH by reducing oxygen to water. So, in the absence of fermentation, oxygen can be used to maintain electron balance, and of course, much more energy is going to be released if we completely burn glucose than if we only partially oxidize glucose. And this is why oxygen allows more energy to be released, or, as you probably learned in high school, more ATP to be produced from glucose metabolism. Now the details of how all of this works are much more complicated than what I have drawn. Obviously fermentation happens the way I drew it, but all the other details about the role of oxygen and how it fits in to these pathways is more complicated than what I've drawn, and we'll cover this in the upcoming lectures. But the key concept is here, and that is the relationship between oxygen and fermentation is really what's illustrated here. These are two different ways that cells can use to dispose of the electron waste that's produced from carbon oxidation and glucose metabolism. Those electrons have to go somewhere. They can go to the product of glycolysis, say pyruvate to lactate, by lactate dehydrogenase, reduce the pyruvate to lactate, fermentation, or they can go somewhere else. A great electron acceptor like oxygen which allows these pathways to happen while saving the pyruvate to do something else, like be completely oxidized to CO2. Now it should be clear here, then, that one of the things that's special about fermentation is that absolutely no oxygen is required to do this, and this is part of the reason why this pathway is so ancient. Fermentation evolved in the pre-oxygen atmosphere, and that really is why it's such an ancient pathway. Only when oxygen levels rose in the atmosphere due to photosynthesis that enough oxygen was around that then other oxidative pathways could evolve as an alternative to fermentation.

~16:57Now, you'll note that the original description of fermentation by Pasteur was ethanol, not lactate. We'll cover the details of ethanol in a minute, but I just want to point out that making ethanol instead of lactate is simply an alternative product to dispose of pyruvate as a more reduced product. So ethanol is a more reduced product than pyruvate in that ethanol generation is an additional adaptation that allows you to dispose of the electron waste while also generating a molecule that's toxic, and therefore, allow organisms that make ethanol to better kill off their neighbors and their environment and compete for resources. But the overall concept is identical. Ethanol is really an alternative to lactate and fermentation but solves exactly the same problem. I want to talk about ethanol metabolism in more detail, but before we do that first I want to go through and just describe at a very high level some of the chemistry that allows all these reactions in glycolysis to work, because you'll see that the chemistry itself is not all that complicated even though we can draw it and it seems like somewhat overwhelming when drawn out as a whole pathway. Now I want to point out, we've already covered the chemistry for a lot of these steps. First of all, there's two dehydrogenase reactions. There's GAPDH and there's lactate dehydrogenase. Here's the mechanism for lactate dehydrogenase, it's a hydride transfer. The same thing happened at GAPDH with the additional details of adding the phosphate. We covered that previously in an earlier lecture. Note, we also covered before the chemistry of pyruvate kinase and phosphoglycerate kinase. In addition, there's two more kinase reactions that exist here, hexokinase and phosphofructokinase. These are really just straightforward phosphotransfer reactions using ATP to transfer a phosphate. Very straightforward, exactly what Professor Yaffe has already talked to you about, about how one does phosphotransfer for kinase reactions on proteins. Now, two other steps are isomerase reactions, triose phosphatase isomerase and glucose phosphate isomerase. We discussed this chemistry back when we did the sugar lectures. This is just acting on the open chain form of the carbohydrate and allows you to interconvert between the ketose and the aldose as I already drew during that lecture. And so that just leaves a few steps left to discuss that we can cover briefly. The first one that we'll talk about is this enolase reaction, which is simply a dehydration. I'll draw it here just to be explicit as to what's going on. So here is 2-phosphoglycerate. Simply dehydration to remove water, and that gives us phosphoenolpyruvate, PEP. The next step I want to discuss, phosphoglycerate mutase, is an example of a mutase reaction that is, remember, it's moving the phosphate from the 3 to the 2 position of glycerate. This is a class of reactions. It has somewhat of an interesting mechanism. And basically precedes by a phosphorylated enzyme intermediate. In the case of phosphoglycerate mutase, this is a histidine, and so there is a histidine with a nitrogen on it in the active site. And effectively, this nitrogen can pick up a phosphate and be primed via a reaction involving a molecule you've seen already from your discussion of hemoglobin with Professor Yaffe, and that's 2,3-bisphosphoglycerate. And so again, here's-- this is glycerate phosphorylated on the 2 and 3 position. So this is 2,3bisphosphoglycerate. And effectively, this molecule can bind in the active site and pick up a phosphate from either of the 2 or the 3 position. I drew it here is picking it up from the 2 position. That would, of course, generate a 3-phosphoglycerate. But more importantly, it ends up with the enzyme having this phosphorylated intermediate in the active site.

~20:39Once it has that phosphorylated intermediate in the active site, now it's ready to catalyze the mutase reaction. So I'll draw here first, this here would be 3-phosphoglycerate. Color the phosphate so you can see what's going on. This would basically, in the active site of the enzyme, pick up the phosphate from the active site and generate transiently this 2,3-bisphosphoglycerate intermediate, and then it can retransfer the phosphate from the other position back on to the active site of the enzyme so it's ready to carry out another catalytic cycle, and in the process, move the phosphate effectively from the 3 position to the 2 position. So you'll see that the phosphate is not actually being-- the same phosphate is not being moved within the same molecule, it's being ping-ponged back and forth off of this phosphate enzyme intermediate, and basically this is how cells catalyze various mutase reactions to move phosphates between hydroxyl groups. And so you have to prime this once with a separately-generated 2,3-bisphosphoglycerate, but once you have that phosphorylated enzyme in the active site, it can now continually ping-pong that phosphate around to effectively convert 3-phosphoglycerate to 2-phosphoglycerate, or an example of another mutase reaction, move the phosphate between two other hydroxyl groups on the same molecule. OK. Now the final reaction that we haven't talked about is this one, the aldolase reaction, which is a really key step in glycolysis because it splits that carbon-carbon bond to take the hexose-- fructose 1,6-bisphosphate, and split it into two trioses, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, which ultimately allows you to make this pathway work. And so I'll take you quickly through how aldolase works. So aldolase also has this amine in the active site. So here's FBP drawn in the open chain form. So you end up with this intermediate in the active site, which can undergo this chemistry to split this into the-- remove the lower half of the molecule. So you can see here, this generates this lower half of the molecule, glyceraldehyde 3-phosphate, as well as the top half, which can regenerate the active site, as well as the other product, dihydroxyacetone phosphate. And so very briefly, that's effectively the chemistry that allows aldolase to carry out this important reaction in glycolysis, splitting that fructose 1,6-bisphosphate into the aldose, glyceraldehyde 3-phosphate, and the ketose dihydroxyacetone phosphate. OK. So that is effectively the chemistry that allows you to turn glucose into pyruvate, as well as pyruvate into lactate, but what about making ethanol? How does that work? Now ethanol is an alternative fermentative product. And so the reactions to make ethanol is basically this. So here's pyruvate. First thing that happens is we lose this CO2. Without a change in oxidation state at the second carbon at the ketone in pyruvate. And so this ketone stays the same oxidation state, it's now an aldehyde. This is a molecule called acetaldehyde. And now we can pick up those two electrons from NADH and reduce this aldehyde to the alcohol, reoxidizing that NADH back to NAD+ and generating ethyl alcohol or ethanol. So, the fermentative part is turning the acetaldehyde into the ethanol because that allows you to dispose of those two electrons, reduce the aldehyde to an alcohol while reoxidizing the NADH to an NAD+, which is what allows glycolysis to continue so that NAD+ is available for the GAPDH step.

~24:01How this reaction works is-- I just drew it. It's exactly how the lactate dehydrogenase works. However, what we also now need to discuss is how you carry out this reaction, this carboxylation to take this CO2 off of pyruvate and make acetaldehyde. Now decarbonization reactions are really common reactions in metabolism. They end up being important for lots of pathways. Obviously if we're going to completely oxidize glucose into CO2, we have to remove CO2 for a molecule, and here's the first one-- first one that we're going to see for how that happens. Now there's two general mechanisms that metabolism uses for decarboxylation reactions, and I want to introduce the concept here of both of them. Now, the first one here is that these are referred to as decarboxylation reactions that occur in the context of either an alpha keto acid or a beta keto acid. Now, what do I mean by that? Well, pyruvate is an alpha keto acid, and that's because here's a acid group. The ketone is alpha to the carboxylic acid. This would be in contrast to this molecule. So I'll just draw with a generic R group. Where here, the ketone is beta to the carboxylic acid-- alpha, beta. So pyruvate is an alpha keto acid. This here is a generic beta keto acid. Now I want to point out that decarboxylations of bait ketone acids are very favorable, and they're favorable for the following reason. And that's because if I remove this CO2 from a beta keto acid, I'm left with this molecule, which hopefully you'll recognize as an enol. And just like we talked about for the pyruvate kinase reaction, enols much prefer to rearrange into the keto form. And so decarbonization of a beta keto acid generates an enol which will rearrange to the ketone. This becomes very favorable, and so it becomes favorable to decarboxylate a beta keto acid. Now, we can't use that exact same chemistry to decarboxylate an alpha keto acid. However, we can help that process, effectively mimicking the same thing that happens with beta keto acid, by instead introducing a cofactor. So I introduced the concept of cofactors when we talked about NAD and NADH. And remember, cofactors are molecules that provide functional groups that help facilitate the chemistry of various reactions. And so there's a cofactor that facilitates the chemistry that allows decarboxylation of alpha keto acids, and that cofactor is abbreviated TPP+, which stands for the thiamine pyrophosphate. TPP+, which is derived from the vitamin thiamine, sometimes also referred to as the vitamin B1. And so this is what TPP+ looks like. OK. So like many of our vitamins or cofactors, it's a complex molecule. So this is vitamin B1 or thiamine, and the cofactor TPP+ is basically the same molecule with a pyrophosphate group added on the end, TPP+. Now, to see what's going on, we're going to focus here just on this part, this reactive part of the molecule, which is right here, which for simplicity, I will just draw like this. So this here is just this basically part of the molecule here, that's the reactive part of TPP+. And what makes this cofactor useful is that this basically can exist in this stabilized carbanion form. This stabilized carbanion can react with alpha keto acid like pyruvate, shown here. And then this intermediate now becomes favorable for decarboxylation of the CO2 that's alpha to the ketone. And then here, we can regenerate the active TPP+ that we started with plus the other product, acetaldehyde. And then that acetaldehyde can then be reduced, regenerating NAD+ and giving alcohol for fermentation. OK.

~27:38Now incidentally, this is how you produce alcohol. How you metabolize alcohol is effectively reversing that last step and further oxidizing the acetaldehyde. And so this is ethanol. So the way ethanol is metabolized. And so the famous alcohol dehydrogenase enzyme basically does that reverse reaction, generates 2 electrons as a hydride ion. They get transferred to NAD+. That NAD+ gets reduced to NADH as this alcohol in ethanol is oxidized to the aldehyde in acetaldehyde. And this can then be further oxidized, that aldehyde to the acid. So here again, two electrons from the hydride iron can reduce NAD+ to NADH. And that generates, in the process, oxidizes the aldehyde to the acid. In this case, this is acetate. The acid form it would be acetic acid, also known as vinegar. And so effectively, the way you turn alcohol into vinegar is by the microorganisms oxidizing the alcohol they produce into acetic acid or vinegar. Now note, this is-- alcohol metabolism is two oxidation reactions. So we talked about last time, oxidation reactions are generally favorable, therefore energy is released. This is why alcohol has calories and it's impossible to make diet alcohol, because it will-- just no way around this. A couple other things about this, as I mentioned. This enzyme catalyzes. This is the famous alcohol dehydrogenase. This enzyme is an aldehyde dehydrogenase. The issue is is that this aldehyde hydrogenate is a less efficient enzyme than alcohol dehydrogenase, thus it can become rate-limiting for alcohol metabolism. And so if you overwhelm the system by drinking too much, effectively this enzyme can't keep up with this enzyme, you generate excess of the toxic acetaldehyde product. Acetaldehyde product is toxic, and this is effectively what generates hangovers.

~29:21It's also the case that there are individuals with polymorphisms of this aldehyde dehydrogenase. This was, I believe, discussed in Professor Yaffe's lectures on enzyme kinetics that have different catalytic efficiency at this step, and the people who have those polymorphisms can't metabolize alcohol as well. They tend to get flushing and whatnot because they build up this toxic acetaldehyde, and in fact, can be quite dangerous if they drink too much. Finally, this also illustrates effectively why when commercially you want to generate alcohol, like in beer or a wine, you need to keep oxygen levels low. Why is that? Because, well, the microbes will read their ethanol. It's a perfectly good source of calories for them as well because of these reactions. But they generate all this electron waste in the process. Those electrons have to go somewhere. Where do they go? Well, oxygen is a great electron acceptor. And so to metabolize this ethanol, they need to put those electrons somewhere. They need to put them on oxygen. And so oxygen is required to carry out this ethanol metabolism by the microorganisms. And so by keeping oxygen out of your fermentation of alcohol in commercial beer and wine production, it prevents the formation of aldehyde and acetic acid products which would be undesirable in your finished drink product. OK. Now this whole discussion also begins to suggest one big way in which glycolysis itself is regulated, and that's effectively by the availability of oxygen. And so to be explicit about this, I want to draw the relationship between fermentation, glycolysis, and oxygen in terms of maintaining electron balance across these pathways.

~30:58And so glucose is, of course, a major fuel for us. It's a major sugar in our blood. Yeast use a lot of glucose from the grapes in their environment. Plants, of course, make glucose as a storage carbohydrate. It's-- we discussed, starch is a polymer of glucose that uses glucose to survive the night. And effectively what they do is turn that glucose into two pyruvate molecules which can be used to generate net 2 ATP as we discussed. But creates this problem that it also generates NADH that needs to be recycled back to NAD+. In the absence of oxygen, you can ferment that pyruvate into lactate or alcohol or some other product that allows you to regenerate NAD+. Or-- and this is a pathway that requires no oxygen. However, that pyruvate can be further oxidized to CO2, but that's going to generate even more electron waste. That electron waste also has to be dealt with, and that requires oxygen or some other place to put those electrons, oxygen to water being a major driver of further oxidative metabolism. Now the beauty of fermentation as a pathway is that no oxygen is needed, but the trade-off is that it's much less efficient. You only get 2 ATP per mole of glucose that's-- 2 moles of ATP per of glucose that's fermented. You can imagine that a lot more energy is released-- if you can further oxidized glucose, you can generate, therefore, a lot more ATP if you use a complete oxidation of glucose, but this requires oxygen. And so a major way that glycolysis and certainly fermentation is regulated is that low oxygen is what promotes fermentation, whereas high oxygen suppresses fermentation, and that makes sense. It's intuitive. You get a lot more energy. If you completely oxidize your glucose, you can more efficiently get energy from oxidation of available reduced glucose carbon if you do it by oxidative pathways, and so therefore, in the presence of plenty of oxygen, you don't need to do fermentation. Whereas if you outstrip oxygen supply, as, say, might happen if your muscles are exercising, burning more ATP than can be kept up with by oxygen delivery from the blood, now you switch over to a more affirmative metabolism, something like lactic acid builds up that can then be dealt with later. Now, this fact that high oxygen can suppress fermentation is experimentally true in many organisms in many contexts. It's likely because the high ATP-ADP that's produced with oxidative glucose metabolism can directly affect some steps or deplete the NAD that's required to drive glycolysis. Exactly the mechanism that causes this is somewhat debated and likely depends on context and conditions. But it also sets out at least a way for us to begin discussing, how is it at a level of individual enzymes or reactions might something like high ATP-ADP ratio effect flux or flow through a pathway? All right. Well, let's just start by talking about what are the two things that could affect flux or flow through the pathway? Well, remember, thermodynamics, delta G, is what determines whether a pathway happens at all. And because delta G is proportional to things like the ATP-ADP ratio, you can imagine if this ratio gets too high, some of the steps might no longer be favorable to occur. That's certainly one possibility. But the other way that regulation can be done is actually kinetic, and this is because even though enzymes don't determine whether a reaction can happen, at least thermodynamically, they oftentimes do determine the rate of the reaction, and when we're talking about rate and flow through-- flux through pathways, flow through pathways, now rate can become very important.

~34:39And so the kinetics of enzymes, exactly what you talked-- heard about with Professor Yaffe, properties like V max or Km of an enzyme, which, of course, can be affected by allostery, and Professor Yaffe covered in great detail, these also, you can imagine, can affect how a pathway is regulated. And of course, things like ATP, ADP, and AMP as small molecules can be good allosteric affectors of some enzymes. And so let's now talk about, if we're going to regulate steps in a pathway, which steps should actually be regulated. And so shown here is, again, coming back to our delta G change map across glycolysis, as we alluded to earlier, the steps where it's hard to go back is this hexokinase step, this phosphofructokinase step, and this pyruvate kinase step, because these are where there's the big change in delta G that now makes it hard to reverse those steps. And it's exactly those steps that are regulated, because that makes sense. If this delta G is thermodynamically driving flux through the pathway, changing the rate of the enzymes that catalyze those steps is also going to change the rate at which the pathway as a whole can happen. Now as a result, it's these steps, hexokinase, phosphofructokinase, pyruvate kinase, that are often referred to as the rate-limiting steps of glycolysis. Personally, I don't like referring to memorizing things like these are the rate-limiting steps of the pathway, because in reality, what step of any pathway is going to depend on the context. However, often this concept of rate limitation when people refer to pathways really is coming from this energetic argument about which steps have the biggest drop in delta G, and it's important for you to understand that, not just memorize what steps are rate-limiting. Now I also want to point out that these steps actually are in interesting places in glycolysis. So this is basically entry into the pathway, and this is exit from the pathway. And this makes sense, too. You don't want to start committing carbon into a pathway unless you're actually going to need it, and you also need to match output to input. And what you'll see is that many steps in pathways that are regulated, in addition to being the ones with the biggest delta G changes, are also often the entrance and exit from the pathway, which, of course, is the case in glycolysis, but also in most pathways it makes a lot of sense just from logical considerations. All right. Now if we're going to control things that the level of enzymes and rate, how can we control rate of a reaction? That is, how can we control enzyme kinetics? Well, if you remember from Professor Yaffe's lectures, you can increase the rate of a reaction-- that is, catalyzed by an enzyme-- by increasing the Vmax of that enzyme; or, if the substrate concentration is close to Km, by lowering the Km of that enzyme. How can you decrease the rate? Of course, you can do the opposite. You can decrease the Vmax; or, if the substrate concentration is close to Km, you can increase the Km. So how can I change these things? How can I alter Vmax or Km of an individual enzyme? Well, there's a number of ways I can do this. One is is I can make more enzyme. What will making more enzyme do? Well, effectively that will increase the Vmax of an enzyme. Or if I get rid of enzyme, that will decrease the Vmax of an enzyme. I can use a different version of the same enzyme. That is, have an enzyme like pyruvate kinase but have it come in a bunch of different varieties, have different genes encode different pyruvate kinase isoforms such that those isoforms have different Vmax and Km relationships with respect to substrate, and effectively, by using a different version of the enzyme, a so-called isoform of an enzyme, you can now have different properties.

~38:38Now you'll note that the change enzyme kinetics this way basically is new transcription translation. That's fairly long on the timescale of needing to adapt to metabolism, but that can work. But sometimes a lion's attacking you, you need to run away quickly, you may not have time to make more enzyme. You also need direct control of the enzymes, and this is where allosteric regulation of enzyme function comes into play. Allosteric regulation can be a result of signaling. Phosphorylate, acetylate an enzyme, that can change its properties. Or you can have metabolite binding. Bind ATP, bind AMP into some allosteric site that can change Vmax or Km and operate on a short time scale to allow metabolism to adapt in a more acute setting. I want to say that time scales matter for real biology if you want to understand physiology and how it applies to how metabolism and physiology relate to biology that you'll encounter in other contexts. You realize that some responses need to be acute. Those allow you to adapt to conditions quickly, be able to get enough ATP to run away from the lion, et cetera. But you also need adaptations that work on longer time scales, because it's things like this that allow you to enact programs that allow you to adapt long-term to whatever new conditions a cell may face in its environment. All right. Now, before I get into talking exactly how these things actually work to regulate glycolysis, I want to discuss a few details about regulation of pathways in general. And while we talked about these with respect to glycolysis, these points will end up coming up over and over again and really apply to other pathways. Now I may not explicitly point this out, but the same considerations for how glycolysis works, how it's regulated has to be true for all metabolic pathways. And remember, any pathway is just variations on relatively few chemical-- chemistry reactions that are repurposed in a way to build some other thing that's useful to the cell, and you'll see this over and over again. Now, it has to be true for every pathway that it has to be thermodynamically favorable, and that means either that the pathway itself, glucose to pyruvate, is thermodynamically favorable, or it's coupled to some kind of energy input, like ATP hydrolysis that allows the pathway to be favorable. Every pathway is going to be built in a way that intermediates are generated along the way that allow that pathway to accomplish its goals. In the case of glycolysis it was the two steps that allowed us to synthesize and incorporate phosphate despite the high ATP-ADP ratio in cells. And in all cases this has to obey the laws of thermodynamics. That is, we have to obey constant conservation of mass. That means we have to account for all carbon, all electrons, delta G has to be less than 0 across the entire pathway. You'll see that the regulated steps will often be those with the largest thermodynamic considerations, like the big drops across hexokinase, phosphofructokinase, and pyruvate kinase. They'll often be at the entrance and exits to pathway because you want to regulate, you don't want to commit something to a pathway if you're not going to use it, and you want to match input and output. And all of this is going to have regulation that acts on both short and long time scales so it allows it to match physiology. All right. Now with that in mind, now let's discuss how we can regulate glycolysis using both short and long time scales as-- using really short time scales, allosteric regulation of glycolysis in a way that makes it most useful to cells. All right. So the first step is uptake of glucose into the cell, which in animals, like us, is passive, but can still be regulated by glucose transporters, often regulated glute proteins. And it's really these glucose transporters that can often determine whether glucose is actually taken up into cells.

~42:39A famous example of this is FDG PET scanning, which is used to measure glucose uptake into tissues in humans, where basically a hydroxyl group is replaced with a positron-emitting fluorine atom, fluorodeoxyglucose, that is taken up and phosphorylated in cells, and it's this lighting up of this on a positron-emitting scan you can tell that which tissues in the body take up glucose and that's a property of cancer, and so it is often used as a way to detect or stage cancer. Another example of this is insulin. Basically one of the major actions of insulin on your tissues is that when glucose is high, your body makes more insulin. That causes glucose transporters to be put on the surface of your muscle and your fat, which allows that glucose now to get into those cells and effectively allows those cells to dispose of the glucose in the blood and keep glucose in the right physiological range. And so uptake of glucose, at least in animals, is one important step that is really controlled largely by whether or not there is a transporter on the surface. All right. Once that's trapped as-- sorry, once it's in the cell as glucose, hexokinase then traps that molecule as glucose 6-phosphate. And it turns out the glucose 6-phosphate is an inhibitor of hexokinase. Why is that? Well, we will see in the next lecture that glucose 6-phosphate can be turned into glycogen in the case of mammals or starch in the case of plants. And so glucose 6-phosphate, if you're going to store glucose, you can ship it off into glycogen, or you could commit it further down into glycolysis. But if you don't need it for a glycolysis and your stores are full, you'll build up glucose 6-phosphate, you want to inhibit hexokinase to keep things from flowing through the system. The next step that's regulated is phosphofructokinase. So phosphofructokinase, of course, makes FTP. The step downstream of that that's regulated is pyruvate kinase, PEP to pyruvate, and it turns out that FTP will stimulate pyruvate kinase, whereas PEP will inhibit phosphofructokinase. Makes sense. You want to match input to output. If you have a lot of FTP, activate the enzyme to dispose of it. If you're building up PEP, tell you to stop sending molecules down the pipeline. It turns out, PFK is actually the major regulatory step of glycolysis, because this is really what commits that carbon to going through glycolysis as opposed to upstream of that, it could be stored or not. So it's really that step that's a commitment step. And so it's also regulated by some of the outputs of the pathway. And so some of those outputs are ATP downstream of pyruvate, which we'll talk about later in the course. There's some other molecules. Alanine, citrate. It turns out that high levels of citrate will also inhibit PFK. High levels of ATP will inhibit phosphofructokinase. Whereas high levels of AMP will activate phosphofructokinase. Makes sense. A major output of glycolysis is to keep the ATP-ADP ratio high, the energy charge high in the cell. If the energy charge is very high, high ATP, shut off phosphofructokinase, don't need to do more glycolysis. If the energy charge low, high AMP, activate phosphofructokinase. If production of other molecules downstream like citrate are high, come back and stop sending things into the pathway. Turns out, alanine can inhibit pyruvate kinase, another step downstream. Great. This is effectively general regulation, allosteric regulation of how glycolysis works. And what I wrote is was generally true across different tissues, but of course, these regulations can be tweaked to be more or less important to match physiology and function. Now I know many of you are going to take the MCAT. And while I would never want to teach just to take a test, a couple of things that you should remember for that test is phosphofructokinase is a major regulator. Its big allosteric regulators are AMP, as well as ATP and citrate.

~46:38Whereas the other steps, hexokinase, you should know that it's regulated by-- negatively regulated by glucose 6phosphate, and pyruvate kinase is positively regulated by FBP. Now one other point that I want to mention, just because it will come up sometimes on MCAT exams, is that there's an additional detail about how PFK is allosterically regulated, and that's because there's this side reaction where fructose 6-phosphate can be phosphorylated on the 2 rather than the 1 position to make this molecule, fructose 2,6-bisphosphate. This is a reaction that is catalyzed by bifunctional enzyme called PFK-2/FBPase. So basically phosphofructokinase on the 2 position, FBPase, we're moving it from the 2 position which allows you to regulate the levels of fructose 2,6-bisphosphate. It turns out that PFK in glycolysis, which, of course, generates FBP, is positively regulated by fructose 2,6-bisphosphate. And the production of fructose 2,6-bisphosphate is something that can be inhibited by ATP, inhibited by phosphoenolpyruvate, and activated by ATP. So exactly what is shown here is allosteric regulation. Just some of this allosteric regulation is via this side enzyme to make a separate product, fructose 2,6-bisphosphate, and there's control theory considerations for why that might happen. All right. So at this point, we've learned how we can start with glucose, oxidase that glucose via a pathway that now allows us to get the energy that cells need to maintain ATP-ADP ratio in a good physiological range despite the high ATP-ADP ratio in cells. However, it should also be clear that in order for this to work, we need a way to actually get that glucose from somewhere to begin with. You probably learned in high school that plants use photosynthesis as a way to get energy from the sun. Well, the synthesis part of photosynthesis is generating the sugar that those plants can then use to burn and keep ATP-ADP high at night and survive the night until the sun comes up again when they can again use energy from the sun directly to get energy. We as animals evolve the way that we have to eat the plants. We have to get the glucose that the plants make, but of course, we also use-- generate-- have the ability to generate some glucose, and this is best illustrated from muscle physiology. And that is, we alluded to earlier, that if we're running away from the lion, our muscles might outstrip the supply of oxygen in the blood and basically generate a whole lot of lactate. Well, that lactate is perfectly good fuel. Just like the yeast, can oxidize or we can reoxidize ethanol. We can also use lactate as a fuel, but it turns out, a big job of our liver is to take that lactate from the blood, as well as other molecules, and regenerate glucose so that our blood glucose remains in a constant level. This cycling of glucose and lactate across the body between the muscle and the liver is sometimes referred to as the Cori cycle and involves a clear set of reactions to turn that lactate back into glucose, a process called gluconeogenesis. And unfortunately we're out of time today, but next time I will start off the lecture by going through how it is that gluconeogenesis works to turn something like lactate back into glucose so that organisms have the glucose to begin with to run glycolysis. Thank you.

2.6肿瘤 Warburg 效应、PKM2 悖论与 FDG-PET 显像：Vander Heiden 教授的癌症代谢前沿课

作为当今国际肿瘤代谢领域的领军人物（2009 年在《Science》发表里程碑综述 Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation），Prof. Matthew Vander Heiden 在 MIT 7.05 课堂上将糖酵解的生化原理与癌症临床诊治及前沿科研做了无缝衔接。这一节不仅是听懂 MIT 7.05 的精华所在，更是近年来各大名校生化考研论述题最青睐的“拔高压轴题”素材！

一、临床肿瘤追踪利器：为什么 ¹⁸F-FDG PET 扫描能精准点亮全身癌灶？

在 L21 课堂谈到葡萄糖跨膜转运（GLUT）与第一步己糖激酶（HK）捕获机制时，Vander Heiden 教授展示了医院肿瘤科每天都在使用的正电子发射断层扫描——¹⁸F-FDG PET 显像（Fluorodeoxyglucose Positron Emission Tomography） 的化学原理：

• 分子结构设计：医生给患者静脉注射的显像剂是 2-氟-2-脱氧-D-葡萄糖（¹⁸F-FDG），即把普通葡萄糖 C2 位上的羟基（-OH）替换成了一个发射正电子的放射性氟同位素原子（¹⁸F）。
• 两步化学陷阱（代谢滞留效应）：

1. 恶性肿瘤细胞为了维持疯狂代谢，细胞膜表面高表达不依赖胰岛素的葡萄糖转运蛋白（GLUT1 / GLUT3），且胞内高表达结合在线粒体外膜上的己糖激酶 II（HK2）。因此，肿瘤组织会像吸尘器一样以远高于正常组织的速度将血液中的 ¹⁸F-FDG 摄取入胞，并在第 1 步被己糖激酶迅速磷酸化为 6-磷酸-2-氟-2-脱氧葡萄糖（¹⁸F-FDG-6-P）——由于带上了磷酸负电荷，它再也无法穿过细胞膜逃回血液 2012·单选！
2. 更绝的是第 2 步：我们在 2.1 节讲过，第 2 步磷酸己糖异构酶（PGI）把醛糖变成酮糖，必须依赖 C2 位的羟基（-OH）形成顺式烯二醇中间体！而 ¹⁸F-FDG-6-P 的 C2 位上是一个强电负性的氟原子（-F）而非羟基，根本无法形成烯二醇中间体，因此反应在第 2 步前被彻底卡死，既不能继续往下走糖酵解，也不能用于合成糖原！

• 结论：¹⁸F-FDG 凭借 GLUT 摄取和己糖激酶磷酸化被加上负电荷锁在胞内，又因缺乏 C2-OH 无法通过磷酸己糖异构酶（PGI）而被迫在肿瘤细胞内大量蓄积，其发射的正电子湮灭信号在 PET 扫描仪上将原发肿瘤与转移灶“精准点亮”（注：正常高耗糖器官如大脑皮层，以及排泄路径如肾脏和膀胱也会显影）。

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二、肿瘤 Warburg 效应（有氧糖酵解）的世纪之谜：为什么癌细胞放着 32 个 ATP 不要，偏要抢糖造乳酸？

1924 年，德国诺贝尔奖得主 奥托·瓦伯格（Otto Warburg） 观察到了一个违背“巴斯德效应”常理的诡异现象：正常分化组织在有氧条件下主要通过线粒体氧化磷酸化将葡萄糖彻底氧化为 CO₂，几乎不产生乳酸；然而，瓦伯格效应（Warburg effect，又称有氧糖酵解 aerobic glycolysis）：指恶性肿瘤细胞（以及快速增殖的胚胎细胞、活化淋巴细胞等）即使在氧气供应极其充足的条件下，依然维持极高的葡萄糖摄取和糖酵解速率，并将绝大部分丙酮酸还原为乳酸排出胞外的代谢重编程现象 ⚑华农高频。

整整八十多年里，人们一直困惑不解：有氧彻底氧化 1 分子葡萄糖能赚 30 或 32 个 ATP，而有氧糖酵解变成乳酸只赚区区 2 个 ATP（效率低了 15 倍！）。难道癌细胞不会算数吗？或者像 Warburg 当年猜测的那样，是因为癌细胞的线粒体坏掉了？

在课堂与科研中，Vander Heiden 教授用铁一般的实验数据推翻了这两个旧假设，给出了震撼学界的真相：

1. 第一，绝大多数肿瘤细胞的线粒体功能完全正常，而且癌细胞根本不缺 ATP！ 切勿误以为肿瘤细胞的线粒体氧化磷酸化完全丧失功能！实际上绝大多数癌细胞线粒体呼吸链完好，且因糖酵解周转速率比氧化磷酸化快 10～100 倍，癌细胞内的 [ATP]/[ADP] 比值长期处于高位，ATP 绝非限制细胞增殖的瓶颈。
2. 第二，限制分裂增殖细胞的真正瓶颈不是 ATP，而是合成新细胞的“砖瓦建材（生物量，Biomass）”和还原力（NADPH）！
   请算一笔细胞分裂的物料账：一个静止的心肌细胞或神经元不需要分裂，它吃进葡萄糖的唯一目的就是榨干每一个电子来造 ATP 维持搏动或电信号；但一个正在迅速分裂的癌细胞（或受精卵、遭遇病原体后克隆扩增的 T 细胞），必须在短短 24 小时内把自己的 DNA、RNA、蛋白质和细胞膜磷脂全部复制翻倍！

• 要造 DNA 和 RNA 的核糖骨架？必须从糖酵解第 1 步产物 G-6-P（以及 F-6-P、G3P）分流进入戊糖磷酸途径（PPP），生成 5-磷酸核糖（R-5-P）和还原力 NADPH 2015/2024·填空；
• 要造细胞膜磷脂的甘油骨架？必须从糖酵解第 4 步拿走磷酸二羟丙酮（DHAP）还原成 3-磷酸甘油（α-磷酸甘油） 2009/2010/2024·单选；
• 要造蛋白质所需的非必需氨基酸和嘌呤/胸腺嘧啶所需的一碳单位（FH₄）？必须从糖酵解第 7 步拿走 3-磷酸甘油酸（3-PG），经磷酸甘油酸脱氢酶（PHGDH）进入丝氨酸（Serine）/甘氨酸与一碳单位合成支路；
• 要造细胞膜的长链脂肪酸和胆固醇？必须用乙酰-CoA 在线粒体合成 柠檬酸 运回胞质裂解 2023/2024/2026·简答！

1. 第三，为什么非要把多余的丙酮酸还原成乳酸排掉？ 如果把海量涌入的丙酮酸全部送进线粒体彻底氧化，会产生天量的 NADH 和 ATP；而我们在 2.1 和 2.5 节刚刚学过，一旦 [ATP]/[ADP] 比值和柠檬酸浓度过高，热力学背压和别构抑制就会立刻把糖酵解的总阀门 PFK-1 死死关上！肿瘤细胞之所以采用有氧糖酵解（Warburg 效应）将大量丙酮酸经 LDHA 还原为乳酸排出，本质上是为了在不触发线粒体高 ATP 反馈抑制的前提下，在细胞质中快速再生 NAD⁺，从而维持极高的糖酵解碳通量，源源不断地为戊糖磷酸途径、丝氨酸合成和脂质合成支路供应生物大分子前体（碳骨架）与还原力（NADPH），满足细胞快速增殖使生物量翻倍的需求！此外，排出的大量乳酸还会酸化肿瘤微环境（降低 pH），帮助癌细胞侵袭胞外基质并抑制抗肿瘤免疫 T 细胞的杀伤活性。

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三、Vander Heiden 教授的成名绝学：为什么癌细胞偏爱低活性的 PKM2？（PKM2 悖论）

更令人拍案叫绝的是糖酵解最后一步——丙酮酸激酶（PK） 在肿瘤中的同工酶切换。人体内的 PKM 基因通过外显子互斥可变剪接（Alternative splicing），可以产生两种仅相差 22 个氨基酸的同工酶：

• PKM1（含外显子 9）：表达于大脑、心肌、骨骼肌等终末分化组织，天然呈 高活性四聚体 构象，不受 F-1,6-BP 别构调节，像一条永不堵车的高速公路一样把 PEP 飞快变成丙酮酸送去氧化供能。
• PKM2（含外显子 10）：几乎所有人类恶性肿瘤细胞和胚胎增殖组织均特异高表达可调节的 PKM2 同工酶，而非组成型高活性的 PKM1！按常理猜想，既然癌细胞糖酵解这么旺盛，难道不应该挑选催化活性更高的 PKM1 吗？恰恰相反！Vander Heiden 教授发现，如果用基因工程强行把癌细胞里的 PKM2 替换成高活性的 PKM1，癌细胞在体内形成肿瘤的能力反而显著下降！

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🎧 MIT 7.05 课堂原声英文实录对照（本节关联 1 讲 · 完整逐段实录）点击展开 / 收起 ▾

🎬 MIT 7.05 Lec 21 · Glycolysis II & Allosteric/Hormonal Regulation⏱ ~50:00

~00:00MITOCW | 21. Glycolysis II/Regulation

~00:01MATTHEW

~00:02OK. Today we will start our discussion of-- with a discussion of glycolysis as a pathway and how we can use

~00:09VANDER

~00:09favorable oxidation of glucose to enable phosphate addition while at the same time producing useful

~00:15HEIDEN:

~00:15intermediates that enable us to do ATP synthase-- ATP synthesis and maintain a high ATP-ADP ratio in cells. Now I drew up here the entire glycolytic pathway, just like you saw at the end of the last lecture, and I want to go through it here step by step. And so the first step in glycolysis, as we already talked about, is we have glucose. This here is, of course, alpha-D-Glucose in the-- I drew it in the alpha form. If the OH was pointing up, that would be the beta form, just some quick review. I'll drop the alpha and beta and the stereochemistry denotion going forward. This glucose molecule, as we discussed previously, is first trapped in cells by being phosphorylated by the enzyme hexokinase, sometimes abbreviated HK, to give glucose 6-phosphate. Not shown here, this glucose 6phosphate then undergoes an isomerase reaction via the open chain form. Again, we discussed this reaction when we discussed sugars. And so this would be catalyzed by glucose phosphate isomerase, and takes it from the aldose to the ketose, and now you have the fructose-- the ketose fructose 6-phosphate shown here in the furanose form. This fructose 6phosphate is then phosphorylated a second time by an enzyme called phosphofructokinase, typically abbreviated PVK. It's phosphorylated on the 1 position to give fructose 1,6-bisphosphate, shown here. That fructose 1,6-bisphosphate can then be cleaved via the enzyme aldolase. Cleavage takes place in the open chain form which I drew up here in the corner. Basically splitting the molecule in half to form two trioses, one of which is the ketose dihydroxyacetone phosphate, basically the top half of the molecule. The other is the bottom half of the molecule, the aldose glyceraldehyde 3-phosphate. These two trioses can be interconverted, the aldose and the ketose via on isomerase. The enzyme that does that is called trioses phosphate isomerase. And moving forward basically takes the dihydroxyacetone phosphate that comes from the top half and can turn all the carbon from fructose 1,6-bisphosphate into two glyceraldehyde 3phosphates. That glyceraldehyde 3-phosphate, as we discussed last time, is the substrate for the GAPDH reaction, the glyceraldehyde 3-phosphate dehydrogenase, GAPDH. which, as we discussed last time, couples oxidation of this aldehyde to this acid with phosphate addition. That oxidation reaction, the electrons have to go somewhere, that's where the cofactor NAD comes in. Picks up two electrons, generates NADH, and in the process, this enzyme generates this molecule, 1,3-bisphosphoglycerate or 1,3-BPG.

~02:41Remember, that 1,3-BPG molecule was one of the useful intermediates whereby we could undergo favorable synthesis of ATP even at the high ATP-ADP ratio found in cells, and that's because we can transfer the phosphate from this acid group onto ADP. That gives us an ATP and the product 3-phosphoglycerate 3-PG carried out by the enzyme phosphoglycerate kinase. That 3-PG molecule can then undergo what's referred to as a mutase reaction. And so the phosphate is moved from the 3 to the 2 position of glycerate. So 3-phosphoglycerate to 2-phosphoglycerate, carried out by phosphoglycerate mutase, often abbreviated PGAM. This 2-phosphoglycerate can then undergo a dehydration to- by an enzyme enolase to give the molecule phosphoenolpyruvate. . Phosphoenolpyruvate was the second of those useful intermediates that allow ATP synthesis despite the high ATP-ADP ratio in cells, and that reaction, as we already discussed, is carried out by pyruvate kinase and allows you to generate another ATP. Now, as I drew out the glycolytic pathway here, I also listed the delta G0 prime for every single step in the reaction. Remember, this is informative as to the equilibrium for each of these individual reactions. You'll note that for many of the steps, that delta G0 prime is positive. Remember what that means. That means that equilibrium favors the left. That is the reverse direction of the entire pathway. However, if you add all of these up, the delta G0 prime for the entire pathway-- so delta G0 prime for the entire pathway, glucose to pyruvate adds up to negative 6.9 kcals per mole. And so that negative 6.9 means that for the entire pathway, equilibrium favors the right. So glucose to pyruvate, the right direction. And that is why this can work as an overall pathway even though some of the individual steps, equilibrium may favor the opposite direction. Now you'll notice that there's several favorable steps where delta G0 prime is quite negative. Pyruvate kinase, phosphoglycerate kinase, hexokinase, phosphofructokinase. As we talked about before, these are able to pull the less favorable steps upstream forward by keeping concentrations of products such as 1,3-bisphosphoglycerate low, thus allowing this net reaction to move forward. You'll notice I drew three steps here as irreversible-- hexokinase, phosphofructokinase, and pyruvate kinase. Remember, these are all reactions in our-- there's no such thing as an irreversible reaction. There are some conditions where the reverse reaction, of course, can happen. Although remember, this-- when we discussed irreversible in metabolism, what we're referring to is irreversible under physiological conditions-- that is, conditions found in cells. You'll notice that those three steps are three of the four steps that have very negative delta G0 prime. And so you would expect that if we were going to reverse those reactions, you would need some kind of energy input, and you'll see later that these are the control points or also regulated steps in this pathway. And there are also the sites, just to give you a preview, that we're going to have to deal with if we're going to try to synthesize glucose, gluconeogenesis, the reverse of glycolysis.

~05:45Now of course, these delta G0 primes are very informative of what happens at equilibrium, but of course, what actually happens in a cell or in any condition, remember, is dependent on the equilibrium constant, but also on the conditions. Remember, it's delta G, delta G equals delta G0 prime plus RT times the log of the products over the reactants. And so delta G is what really matters for any individual reactions, and what's shown here on this slide is basically a approximate delta G0 change across the glycolytic pathway based on someone's approximation of the conditions found in cells. Now we can see and looking at this that there's really three big drops in delta G across the pathway. Here's the hexokinase reaction, here's the phosphofructokinase reaction, here's the pyruvate kinase reaction. And so it makes sense that these would be, then, the irreversible steps because there is what is actually pulling the pathway forward. It also makes sense that one would exert control at each of these steps, because this is what drives the flux of the pathway forward, but it's also where it's difficult to go back. Once you get here on this flat part of the curve energetically it doesn't matter so much which direction you go along the curve, but to try to climb back up each of these hills becomes difficult. And so we'll revisit this later in the lecture, but this is basically where the control points will end up happening in this pathway. Now, I want to note a couple other features about this pathway, and that is that we had to invest some energy early. You see you cost an ATP at the hexokinase and the phosphofructokinase step and then you harvest that ATP much later at the phosphoglycerate kinase and the pyruvate kinase steps. The textbook typically will split glycolysis into what they refer to as two stages, an investment stage and a harvesting stage, and that basically relates to that fact. And that's because you have a glucose molecule that then you use to generate two trioses. And then each of those trioses can be used to generate a pyruvate molecule. And so it costs 2 ATP to generate those to trioses, but then each triose generated allows you to recapture 2 ATPs. And so what that means, then, is that the net output from glycolysis is 2 ATPs. And so glucose going to two glyceraldehyde 3-phosphates costs you, whereas using those two glyceraldehyde 3-phosphates to generate two pyruvates allows you to recover 4 ATP for a net of plus 2 ATP molecules produced per glucose metabolized. Now if we add up the entire stoichiometry of what's going on here, we can also draw it another way, and that is we can basically have glucose plus 2 ATP molecules plus 2 inorganic phosphates, goes to 2 pyruvates plus 2 ATP, and that's because we're able to incorporate 2 phosphate molecules here at the GAPDH step. Now that's great, but if you're paying attention, you notice that we're missing one other thing in this stoichiometry, and that's that cofactor NAD that was also required to maintain electron balance across the GAPDH step. And so remember that for these pathways to work, we have to balance all things that are going in with all things are going out-- we can't create or destroy matter. And so those electrons have to be dealt with. When we discussed the GAPDH reaction last time, we needed that cofactor NAD in order to act as an acceptor for the electrons to allow that oxidative phosphorylation, if you will, of glyceraldehyde 3-phosphate to 1,3bisphosphoglycerate.

~09:22And so in order for this pathway to work, we need a way to regenerate that NADH back to an NAD in order to make electron balance. In other words, we have to deal with the fact that we've generated-- or that we required 2 NAD+ to run this pathway and generate 2 NADH. And so we need a way to get rid of those electrons from NADH to regenerate that NAD and allow the pathway to be balanced. Now it should be clear that this has to happen stoichiometrically for every single glucose molecule that goes through glycolysis. That is, we have to regenerate 2 NAD plusses for every glucose that we're going to convert into 2 pyruvates. And so for this NADH reoxidation to occur, we need a place to transfer those two electron pairs. And so just like any oxidation and reduction reaction, if we're going to oxidize NADH back to NAD+, something else has to be reduced, and that thing has to be reduced stoichiometrically with every single pyruvate that's produced from glucose and glycolysis. And effectively you will see that this is the role of fermentation. It's an ability for us to solve this redox problem, that we have to dispose of those electrons in the oxidation at the GAPDH step in order to deal with this electron waste of the pathway. Now, the useful way to do this is that we're generating a pyruvate stoichiometrically. And so we can use that stoichiometrically, that product pyruvate by reducing it to form the waste product, and that's effectively what fermentation is. It's turning that pyruvate into ethanol or lactate, which is reducing that pyruvate, picking up the electron waste from the NADH, and allowing you to regenerate the NAD that's required to run the GAPDH step. And so I'll just illustrate this. The simplest fermentation reaction that we can do is the run that's done by animals, and that's to generate lactate. And so to just illustrate this explicitly, here is-- so this here would be the reduced form of NAD, NADH. So this here this-- this is the nicotinamide group. Remember, it's hooked up to an ADP ribose to give nicotinamide adenine dinucleotide in the reduced form. So this can transfer electrons to pyruvate. Here's pyruvate. So as we showed last time, we can take 2 electrons from NADH reduced. That gives us this hydride ion, which can generate lactate. Plus, what we're left with is the oxidized form of the cofactor NAD+. And now this NAD+ can be reduced back to NADH at the GAPDH step, and the electrons from NADH, as it's being reoxidized to NAD, can go and take pyruvate and reduce it to lactate. So NAD gets reoxidized to NAD. Pyruvate now gets-- the ketone gets reduced to the alcohol as you go from pyruvate to lactate. Opposite of what's going on in the GAPDH step. There, the aldehyde is oxidized to the acid, while NAD+ oxidized goes-- is reduced to NADH. Here, NADH is reoxidized to NAD while taking the pyruvate and re-reducing it to lactate. This, by adding here, allows us now to regain balance across the entire pathway. This step is catalyzed by the enzyme lactate dehydrogenase, often abbreviated LDH. And that allows regenerating the NAD+ that's needed for the GAPDH step, which allows us to deal with the stoichiometry problem of the entire pathway. And so what fermentation does for cells is allows electron disposal by taking the product of the pathway pyruvate, reducing it stoichiometrically to form a reduced product-- as shown here, lactate, and that allows you to maintain electron balance across the entire pathway. And so this entire fermentation pathway, glucose to lactate, now allows the generation of the net generation of 2 ATP and run this pathway in cells so cells can maintain a high ATP-ADP ratio by fermenting glucose into lactate.

~13:14Now, if you're paying attention, this also now begins to describe why it is that oxygen is such a useful molecule for metabolism and actually why it's quite key to supporting the bioenergetics of cells. And so recall, oxygen was there when we burned wood and we used that example. And why oxygen is so important for burning wood, why it's important for doing glucose catabolism in our cells is that oxygen is a really great electron acceptor. That is, I can draw out oxygen can take 2 electrons that are generated as NADH waste, if you want to call it that, plus protons, and this generates water. And so an alternative to fermentation in order to allow glucose oxidation is rather than do fermentation, is to transfer those electrons to oxygen to run the pathway another way. In other words, if we write glucose to pyruvate and realize that this is an oxidation reaction, and so therefore, generates electron waste in the form of NADH that has to be recycled to NAD, we can do so by putting those reoxidizing NADH, means something else has to be reduced. This something else could be oxygen as a good electron acceptor to be reduced to water and find an alternative place to put those electrons. Or, if oxygen is not present, now we can do fermentation, turning pyruvate into lactate. It should be clear that if we want to completely oxidize glucose, as in burning wood-- that is, turn it-- all the carbon and glucose into the most oxidized form of carbon, CO2, this also requires places to put electrons. That is, we could generate more NADH. And of course, each of those electrons have to be dealt with. And the final home for those electrons needs to be something that's a good electron acceptor like oxygen, allowing you to continually reoxidize your NADH by reducing oxygen to water. So, in the absence of fermentation, oxygen can be used to maintain electron balance, and of course, much more energy is going to be released if we completely burn glucose than if we only partially oxidize glucose. And this is why oxygen allows more energy to be released, or, as you probably learned in high school, more ATP to be produced from glucose metabolism. Now the details of how all of this works are much more complicated than what I have drawn. Obviously fermentation happens the way I drew it, but all the other details about the role of oxygen and how it fits in to these pathways is more complicated than what I've drawn, and we'll cover this in the upcoming lectures. But the key concept is here, and that is the relationship between oxygen and fermentation is really what's illustrated here. These are two different ways that cells can use to dispose of the electron waste that's produced from carbon oxidation and glucose metabolism. Those electrons have to go somewhere. They can go to the product of glycolysis, say pyruvate to lactate, by lactate dehydrogenase, reduce the pyruvate to lactate, fermentation, or they can go somewhere else. A great electron acceptor like oxygen which allows these pathways to happen while saving the pyruvate to do something else, like be completely oxidized to CO2. Now it should be clear here, then, that one of the things that's special about fermentation is that absolutely no oxygen is required to do this, and this is part of the reason why this pathway is so ancient. Fermentation evolved in the pre-oxygen atmosphere, and that really is why it's such an ancient pathway. Only when oxygen levels rose in the atmosphere due to photosynthesis that enough oxygen was around that then other oxidative pathways could evolve as an alternative to fermentation.

~16:57Now, you'll note that the original description of fermentation by Pasteur was ethanol, not lactate. We'll cover the details of ethanol in a minute, but I just want to point out that making ethanol instead of lactate is simply an alternative product to dispose of pyruvate as a more reduced product. So ethanol is a more reduced product than pyruvate in that ethanol generation is an additional adaptation that allows you to dispose of the electron waste while also generating a molecule that's toxic, and therefore, allow organisms that make ethanol to better kill off their neighbors and their environment and compete for resources. But the overall concept is identical. Ethanol is really an alternative to lactate and fermentation but solves exactly the same problem. I want to talk about ethanol metabolism in more detail, but before we do that first I want to go through and just describe at a very high level some of the chemistry that allows all these reactions in glycolysis to work, because you'll see that the chemistry itself is not all that complicated even though we can draw it and it seems like somewhat overwhelming when drawn out as a whole pathway. Now I want to point out, we've already covered the chemistry for a lot of these steps. First of all, there's two dehydrogenase reactions. There's GAPDH and there's lactate dehydrogenase. Here's the mechanism for lactate dehydrogenase, it's a hydride transfer. The same thing happened at GAPDH with the additional details of adding the phosphate. We covered that previously in an earlier lecture. Note, we also covered before the chemistry of pyruvate kinase and phosphoglycerate kinase. In addition, there's two more kinase reactions that exist here, hexokinase and phosphofructokinase. These are really just straightforward phosphotransfer reactions using ATP to transfer a phosphate. Very straightforward, exactly what Professor Yaffe has already talked to you about, about how one does phosphotransfer for kinase reactions on proteins. Now, two other steps are isomerase reactions, triose phosphatase isomerase and glucose phosphate isomerase. We discussed this chemistry back when we did the sugar lectures. This is just acting on the open chain form of the carbohydrate and allows you to interconvert between the ketose and the aldose as I already drew during that lecture. And so that just leaves a few steps left to discuss that we can cover briefly. The first one that we'll talk about is this enolase reaction, which is simply a dehydration. I'll draw it here just to be explicit as to what's going on. So here is 2-phosphoglycerate. Simply dehydration to remove water, and that gives us phosphoenolpyruvate, PEP. The next step I want to discuss, phosphoglycerate mutase, is an example of a mutase reaction that is, remember, it's moving the phosphate from the 3 to the 2 position of glycerate. This is a class of reactions. It has somewhat of an interesting mechanism. And basically precedes by a phosphorylated enzyme intermediate. In the case of phosphoglycerate mutase, this is a histidine, and so there is a histidine with a nitrogen on it in the active site. And effectively, this nitrogen can pick up a phosphate and be primed via a reaction involving a molecule you've seen already from your discussion of hemoglobin with Professor Yaffe, and that's 2,3-bisphosphoglycerate. And so again, here's-- this is glycerate phosphorylated on the 2 and 3 position. So this is 2,3bisphosphoglycerate. And effectively, this molecule can bind in the active site and pick up a phosphate from either of the 2 or the 3 position. I drew it here is picking it up from the 2 position. That would, of course, generate a 3-phosphoglycerate. But more importantly, it ends up with the enzyme having this phosphorylated intermediate in the active site.

~20:39Once it has that phosphorylated intermediate in the active site, now it's ready to catalyze the mutase reaction. So I'll draw here first, this here would be 3-phosphoglycerate. Color the phosphate so you can see what's going on. This would basically, in the active site of the enzyme, pick up the phosphate from the active site and generate transiently this 2,3-bisphosphoglycerate intermediate, and then it can retransfer the phosphate from the other position back on to the active site of the enzyme so it's ready to carry out another catalytic cycle, and in the process, move the phosphate effectively from the 3 position to the 2 position. So you'll see that the phosphate is not actually being-- the same phosphate is not being moved within the same molecule, it's being ping-ponged back and forth off of this phosphate enzyme intermediate, and basically this is how cells catalyze various mutase reactions to move phosphates between hydroxyl groups. And so you have to prime this once with a separately-generated 2,3-bisphosphoglycerate, but once you have that phosphorylated enzyme in the active site, it can now continually ping-pong that phosphate around to effectively convert 3-phosphoglycerate to 2-phosphoglycerate, or an example of another mutase reaction, move the phosphate between two other hydroxyl groups on the same molecule. OK. Now the final reaction that we haven't talked about is this one, the aldolase reaction, which is a really key step in glycolysis because it splits that carbon-carbon bond to take the hexose-- fructose 1,6-bisphosphate, and split it into two trioses, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, which ultimately allows you to make this pathway work. And so I'll take you quickly through how aldolase works. So aldolase also has this amine in the active site. So here's FBP drawn in the open chain form. So you end up with this intermediate in the active site, which can undergo this chemistry to split this into the-- remove the lower half of the molecule. So you can see here, this generates this lower half of the molecule, glyceraldehyde 3-phosphate, as well as the top half, which can regenerate the active site, as well as the other product, dihydroxyacetone phosphate. And so very briefly, that's effectively the chemistry that allows aldolase to carry out this important reaction in glycolysis, splitting that fructose 1,6-bisphosphate into the aldose, glyceraldehyde 3-phosphate, and the ketose dihydroxyacetone phosphate. OK. So that is effectively the chemistry that allows you to turn glucose into pyruvate, as well as pyruvate into lactate, but what about making ethanol? How does that work? Now ethanol is an alternative fermentative product. And so the reactions to make ethanol is basically this. So here's pyruvate. First thing that happens is we lose this CO2. Without a change in oxidation state at the second carbon at the ketone in pyruvate. And so this ketone stays the same oxidation state, it's now an aldehyde. This is a molecule called acetaldehyde. And now we can pick up those two electrons from NADH and reduce this aldehyde to the alcohol, reoxidizing that NADH back to NAD+ and generating ethyl alcohol or ethanol. So, the fermentative part is turning the acetaldehyde into the ethanol because that allows you to dispose of those two electrons, reduce the aldehyde to an alcohol while reoxidizing the NADH to an NAD+, which is what allows glycolysis to continue so that NAD+ is available for the GAPDH step.

~24:01How this reaction works is-- I just drew it. It's exactly how the lactate dehydrogenase works. However, what we also now need to discuss is how you carry out this reaction, this carboxylation to take this CO2 off of pyruvate and make acetaldehyde. Now decarbonization reactions are really common reactions in metabolism. They end up being important for lots of pathways. Obviously if we're going to completely oxidize glucose into CO2, we have to remove CO2 for a molecule, and here's the first one-- first one that we're going to see for how that happens. Now there's two general mechanisms that metabolism uses for decarboxylation reactions, and I want to introduce the concept here of both of them. Now, the first one here is that these are referred to as decarboxylation reactions that occur in the context of either an alpha keto acid or a beta keto acid. Now, what do I mean by that? Well, pyruvate is an alpha keto acid, and that's because here's a acid group. The ketone is alpha to the carboxylic acid. This would be in contrast to this molecule. So I'll just draw with a generic R group. Where here, the ketone is beta to the carboxylic acid-- alpha, beta. So pyruvate is an alpha keto acid. This here is a generic beta keto acid. Now I want to point out that decarboxylations of bait ketone acids are very favorable, and they're favorable for the following reason. And that's because if I remove this CO2 from a beta keto acid, I'm left with this molecule, which hopefully you'll recognize as an enol. And just like we talked about for the pyruvate kinase reaction, enols much prefer to rearrange into the keto form. And so decarbonization of a beta keto acid generates an enol which will rearrange to the ketone. This becomes very favorable, and so it becomes favorable to decarboxylate a beta keto acid. Now, we can't use that exact same chemistry to decarboxylate an alpha keto acid. However, we can help that process, effectively mimicking the same thing that happens with beta keto acid, by instead introducing a cofactor. So I introduced the concept of cofactors when we talked about NAD and NADH. And remember, cofactors are molecules that provide functional groups that help facilitate the chemistry of various reactions. And so there's a cofactor that facilitates the chemistry that allows decarboxylation of alpha keto acids, and that cofactor is abbreviated TPP+, which stands for the thiamine pyrophosphate. TPP+, which is derived from the vitamin thiamine, sometimes also referred to as the vitamin B1. And so this is what TPP+ looks like. OK. So like many of our vitamins or cofactors, it's a complex molecule. So this is vitamin B1 or thiamine, and the cofactor TPP+ is basically the same molecule with a pyrophosphate group added on the end, TPP+. Now, to see what's going on, we're going to focus here just on this part, this reactive part of the molecule, which is right here, which for simplicity, I will just draw like this. So this here is just this basically part of the molecule here, that's the reactive part of TPP+. And what makes this cofactor useful is that this basically can exist in this stabilized carbanion form. This stabilized carbanion can react with alpha keto acid like pyruvate, shown here. And then this intermediate now becomes favorable for decarboxylation of the CO2 that's alpha to the ketone. And then here, we can regenerate the active TPP+ that we started with plus the other product, acetaldehyde. And then that acetaldehyde can then be reduced, regenerating NAD+ and giving alcohol for fermentation. OK.

~27:38Now incidentally, this is how you produce alcohol. How you metabolize alcohol is effectively reversing that last step and further oxidizing the acetaldehyde. And so this is ethanol. So the way ethanol is metabolized. And so the famous alcohol dehydrogenase enzyme basically does that reverse reaction, generates 2 electrons as a hydride ion. They get transferred to NAD+. That NAD+ gets reduced to NADH as this alcohol in ethanol is oxidized to the aldehyde in acetaldehyde. And this can then be further oxidized, that aldehyde to the acid. So here again, two electrons from the hydride iron can reduce NAD+ to NADH. And that generates, in the process, oxidizes the aldehyde to the acid. In this case, this is acetate. The acid form it would be acetic acid, also known as vinegar. And so effectively, the way you turn alcohol into vinegar is by the microorganisms oxidizing the alcohol they produce into acetic acid or vinegar. Now note, this is-- alcohol metabolism is two oxidation reactions. So we talked about last time, oxidation reactions are generally favorable, therefore energy is released. This is why alcohol has calories and it's impossible to make diet alcohol, because it will-- just no way around this. A couple other things about this, as I mentioned. This enzyme catalyzes. This is the famous alcohol dehydrogenase. This enzyme is an aldehyde dehydrogenase. The issue is is that this aldehyde hydrogenate is a less efficient enzyme than alcohol dehydrogenase, thus it can become rate-limiting for alcohol metabolism. And so if you overwhelm the system by drinking too much, effectively this enzyme can't keep up with this enzyme, you generate excess of the toxic acetaldehyde product. Acetaldehyde product is toxic, and this is effectively what generates hangovers.

~29:21It's also the case that there are individuals with polymorphisms of this aldehyde dehydrogenase. This was, I believe, discussed in Professor Yaffe's lectures on enzyme kinetics that have different catalytic efficiency at this step, and the people who have those polymorphisms can't metabolize alcohol as well. They tend to get flushing and whatnot because they build up this toxic acetaldehyde, and in fact, can be quite dangerous if they drink too much. Finally, this also illustrates effectively why when commercially you want to generate alcohol, like in beer or a wine, you need to keep oxygen levels low. Why is that? Because, well, the microbes will read their ethanol. It's a perfectly good source of calories for them as well because of these reactions. But they generate all this electron waste in the process. Those electrons have to go somewhere. Where do they go? Well, oxygen is a great electron acceptor. And so to metabolize this ethanol, they need to put those electrons somewhere. They need to put them on oxygen. And so oxygen is required to carry out this ethanol metabolism by the microorganisms. And so by keeping oxygen out of your fermentation of alcohol in commercial beer and wine production, it prevents the formation of aldehyde and acetic acid products which would be undesirable in your finished drink product. OK. Now this whole discussion also begins to suggest one big way in which glycolysis itself is regulated, and that's effectively by the availability of oxygen. And so to be explicit about this, I want to draw the relationship between fermentation, glycolysis, and oxygen in terms of maintaining electron balance across these pathways.

~30:58And so glucose is, of course, a major fuel for us. It's a major sugar in our blood. Yeast use a lot of glucose from the grapes in their environment. Plants, of course, make glucose as a storage carbohydrate. It's-- we discussed, starch is a polymer of glucose that uses glucose to survive the night. And effectively what they do is turn that glucose into two pyruvate molecules which can be used to generate net 2 ATP as we discussed. But creates this problem that it also generates NADH that needs to be recycled back to NAD+. In the absence of oxygen, you can ferment that pyruvate into lactate or alcohol or some other product that allows you to regenerate NAD+. Or-- and this is a pathway that requires no oxygen. However, that pyruvate can be further oxidized to CO2, but that's going to generate even more electron waste. That electron waste also has to be dealt with, and that requires oxygen or some other place to put those electrons, oxygen to water being a major driver of further oxidative metabolism. Now the beauty of fermentation as a pathway is that no oxygen is needed, but the trade-off is that it's much less efficient. You only get 2 ATP per mole of glucose that's-- 2 moles of ATP per of glucose that's fermented. You can imagine that a lot more energy is released-- if you can further oxidized glucose, you can generate, therefore, a lot more ATP if you use a complete oxidation of glucose, but this requires oxygen. And so a major way that glycolysis and certainly fermentation is regulated is that low oxygen is what promotes fermentation, whereas high oxygen suppresses fermentation, and that makes sense. It's intuitive. You get a lot more energy. If you completely oxidize your glucose, you can more efficiently get energy from oxidation of available reduced glucose carbon if you do it by oxidative pathways, and so therefore, in the presence of plenty of oxygen, you don't need to do fermentation. Whereas if you outstrip oxygen supply, as, say, might happen if your muscles are exercising, burning more ATP than can be kept up with by oxygen delivery from the blood, now you switch over to a more affirmative metabolism, something like lactic acid builds up that can then be dealt with later. Now, this fact that high oxygen can suppress fermentation is experimentally true in many organisms in many contexts. It's likely because the high ATP-ADP that's produced with oxidative glucose metabolism can directly affect some steps or deplete the NAD that's required to drive glycolysis. Exactly the mechanism that causes this is somewhat debated and likely depends on context and conditions. But it also sets out at least a way for us to begin discussing, how is it at a level of individual enzymes or reactions might something like high ATP-ADP ratio effect flux or flow through a pathway? All right. Well, let's just start by talking about what are the two things that could affect flux or flow through the pathway? Well, remember, thermodynamics, delta G, is what determines whether a pathway happens at all. And because delta G is proportional to things like the ATP-ADP ratio, you can imagine if this ratio gets too high, some of the steps might no longer be favorable to occur. That's certainly one possibility. But the other way that regulation can be done is actually kinetic, and this is because even though enzymes don't determine whether a reaction can happen, at least thermodynamically, they oftentimes do determine the rate of the reaction, and when we're talking about rate and flow through-- flux through pathways, flow through pathways, now rate can become very important.

~34:39And so the kinetics of enzymes, exactly what you talked-- heard about with Professor Yaffe, properties like V max or Km of an enzyme, which, of course, can be affected by allostery, and Professor Yaffe covered in great detail, these also, you can imagine, can affect how a pathway is regulated. And of course, things like ATP, ADP, and AMP as small molecules can be good allosteric affectors of some enzymes. And so let's now talk about, if we're going to regulate steps in a pathway, which steps should actually be regulated. And so shown here is, again, coming back to our delta G change map across glycolysis, as we alluded to earlier, the steps where it's hard to go back is this hexokinase step, this phosphofructokinase step, and this pyruvate kinase step, because these are where there's the big change in delta G that now makes it hard to reverse those steps. And it's exactly those steps that are regulated, because that makes sense. If this delta G is thermodynamically driving flux through the pathway, changing the rate of the enzymes that catalyze those steps is also going to change the rate at which the pathway as a whole can happen. Now as a result, it's these steps, hexokinase, phosphofructokinase, pyruvate kinase, that are often referred to as the rate-limiting steps of glycolysis. Personally, I don't like referring to memorizing things like these are the rate-limiting steps of the pathway, because in reality, what step of any pathway is going to depend on the context. However, often this concept of rate limitation when people refer to pathways really is coming from this energetic argument about which steps have the biggest drop in delta G, and it's important for you to understand that, not just memorize what steps are rate-limiting. Now I also want to point out that these steps actually are in interesting places in glycolysis. So this is basically entry into the pathway, and this is exit from the pathway. And this makes sense, too. You don't want to start committing carbon into a pathway unless you're actually going to need it, and you also need to match output to input. And what you'll see is that many steps in pathways that are regulated, in addition to being the ones with the biggest delta G changes, are also often the entrance and exit from the pathway, which, of course, is the case in glycolysis, but also in most pathways it makes a lot of sense just from logical considerations. All right. Now if we're going to control things that the level of enzymes and rate, how can we control rate of a reaction? That is, how can we control enzyme kinetics? Well, if you remember from Professor Yaffe's lectures, you can increase the rate of a reaction-- that is, catalyzed by an enzyme-- by increasing the Vmax of that enzyme; or, if the substrate concentration is close to Km, by lowering the Km of that enzyme. How can you decrease the rate? Of course, you can do the opposite. You can decrease the Vmax; or, if the substrate concentration is close to Km, you can increase the Km. So how can I change these things? How can I alter Vmax or Km of an individual enzyme? Well, there's a number of ways I can do this. One is is I can make more enzyme. What will making more enzyme do? Well, effectively that will increase the Vmax of an enzyme. Or if I get rid of enzyme, that will decrease the Vmax of an enzyme. I can use a different version of the same enzyme. That is, have an enzyme like pyruvate kinase but have it come in a bunch of different varieties, have different genes encode different pyruvate kinase isoforms such that those isoforms have different Vmax and Km relationships with respect to substrate, and effectively, by using a different version of the enzyme, a so-called isoform of an enzyme, you can now have different properties.

~38:38Now you'll note that the change enzyme kinetics this way basically is new transcription translation. That's fairly long on the timescale of needing to adapt to metabolism, but that can work. But sometimes a lion's attacking you, you need to run away quickly, you may not have time to make more enzyme. You also need direct control of the enzymes, and this is where allosteric regulation of enzyme function comes into play. Allosteric regulation can be a result of signaling. Phosphorylate, acetylate an enzyme, that can change its properties. Or you can have metabolite binding. Bind ATP, bind AMP into some allosteric site that can change Vmax or Km and operate on a short time scale to allow metabolism to adapt in a more acute setting. I want to say that time scales matter for real biology if you want to understand physiology and how it applies to how metabolism and physiology relate to biology that you'll encounter in other contexts. You realize that some responses need to be acute. Those allow you to adapt to conditions quickly, be able to get enough ATP to run away from the lion, et cetera. But you also need adaptations that work on longer time scales, because it's things like this that allow you to enact programs that allow you to adapt long-term to whatever new conditions a cell may face in its environment. All right. Now, before I get into talking exactly how these things actually work to regulate glycolysis, I want to discuss a few details about regulation of pathways in general. And while we talked about these with respect to glycolysis, these points will end up coming up over and over again and really apply to other pathways. Now I may not explicitly point this out, but the same considerations for how glycolysis works, how it's regulated has to be true for all metabolic pathways. And remember, any pathway is just variations on relatively few chemical-- chemistry reactions that are repurposed in a way to build some other thing that's useful to the cell, and you'll see this over and over again. Now, it has to be true for every pathway that it has to be thermodynamically favorable, and that means either that the pathway itself, glucose to pyruvate, is thermodynamically favorable, or it's coupled to some kind of energy input, like ATP hydrolysis that allows the pathway to be favorable. Every pathway is going to be built in a way that intermediates are generated along the way that allow that pathway to accomplish its goals. In the case of glycolysis it was the two steps that allowed us to synthesize and incorporate phosphate despite the high ATP-ADP ratio in cells. And in all cases this has to obey the laws of thermodynamics. That is, we have to obey constant conservation of mass. That means we have to account for all carbon, all electrons, delta G has to be less than 0 across the entire pathway. You'll see that the regulated steps will often be those with the largest thermodynamic considerations, like the big drops across hexokinase, phosphofructokinase, and pyruvate kinase. They'll often be at the entrance and exits to pathway because you want to regulate, you don't want to commit something to a pathway if you're not going to use it, and you want to match input and output. And all of this is going to have regulation that acts on both short and long time scales so it allows it to match physiology. All right. Now with that in mind, now let's discuss how we can regulate glycolysis using both short and long time scales as-- using really short time scales, allosteric regulation of glycolysis in a way that makes it most useful to cells. All right. So the first step is uptake of glucose into the cell, which in animals, like us, is passive, but can still be regulated by glucose transporters, often regulated glute proteins. And it's really these glucose transporters that can often determine whether glucose is actually taken up into cells.

~42:39A famous example of this is FDG PET scanning, which is used to measure glucose uptake into tissues in humans, where basically a hydroxyl group is replaced with a positron-emitting fluorine atom, fluorodeoxyglucose, that is taken up and phosphorylated in cells, and it's this lighting up of this on a positron-emitting scan you can tell that which tissues in the body take up glucose and that's a property of cancer, and so it is often used as a way to detect or stage cancer. Another example of this is insulin. Basically one of the major actions of insulin on your tissues is that when glucose is high, your body makes more insulin. That causes glucose transporters to be put on the surface of your muscle and your fat, which allows that glucose now to get into those cells and effectively allows those cells to dispose of the glucose in the blood and keep glucose in the right physiological range. And so uptake of glucose, at least in animals, is one important step that is really controlled largely by whether or not there is a transporter on the surface. All right. Once that's trapped as-- sorry, once it's in the cell as glucose, hexokinase then traps that molecule as glucose 6-phosphate. And it turns out the glucose 6-phosphate is an inhibitor of hexokinase. Why is that? Well, we will see in the next lecture that glucose 6-phosphate can be turned into glycogen in the case of mammals or starch in the case of plants. And so glucose 6-phosphate, if you're going to store glucose, you can ship it off into glycogen, or you could commit it further down into glycolysis. But if you don't need it for a glycolysis and your stores are full, you'll build up glucose 6-phosphate, you want to inhibit hexokinase to keep things from flowing through the system. The next step that's regulated is phosphofructokinase. So phosphofructokinase, of course, makes FTP. The step downstream of that that's regulated is pyruvate kinase, PEP to pyruvate, and it turns out that FTP will stimulate pyruvate kinase, whereas PEP will inhibit phosphofructokinase. Makes sense. You want to match input to output. If you have a lot of FTP, activate the enzyme to dispose of it. If you're building up PEP, tell you to stop sending molecules down the pipeline. It turns out, PFK is actually the major regulatory step of glycolysis, because this is really what commits that carbon to going through glycolysis as opposed to upstream of that, it could be stored or not. So it's really that step that's a commitment step. And so it's also regulated by some of the outputs of the pathway. And so some of those outputs are ATP downstream of pyruvate, which we'll talk about later in the course. There's some other molecules. Alanine, citrate. It turns out that high levels of citrate will also inhibit PFK. High levels of ATP will inhibit phosphofructokinase. Whereas high levels of AMP will activate phosphofructokinase. Makes sense. A major output of glycolysis is to keep the ATP-ADP ratio high, the energy charge high in the cell. If the energy charge is very high, high ATP, shut off phosphofructokinase, don't need to do more glycolysis. If the energy charge low, high AMP, activate phosphofructokinase. If production of other molecules downstream like citrate are high, come back and stop sending things into the pathway. Turns out, alanine can inhibit pyruvate kinase, another step downstream. Great. This is effectively general regulation, allosteric regulation of how glycolysis works. And what I wrote is was generally true across different tissues, but of course, these regulations can be tweaked to be more or less important to match physiology and function. Now I know many of you are going to take the MCAT. And while I would never want to teach just to take a test, a couple of things that you should remember for that test is phosphofructokinase is a major regulator. Its big allosteric regulators are AMP, as well as ATP and citrate.

~46:38Whereas the other steps, hexokinase, you should know that it's regulated by-- negatively regulated by glucose 6phosphate, and pyruvate kinase is positively regulated by FBP. Now one other point that I want to mention, just because it will come up sometimes on MCAT exams, is that there's an additional detail about how PFK is allosterically regulated, and that's because there's this side reaction where fructose 6-phosphate can be phosphorylated on the 2 rather than the 1 position to make this molecule, fructose 2,6-bisphosphate. This is a reaction that is catalyzed by bifunctional enzyme called PFK-2/FBPase. So basically phosphofructokinase on the 2 position, FBPase, we're moving it from the 2 position which allows you to regulate the levels of fructose 2,6-bisphosphate. It turns out that PFK in glycolysis, which, of course, generates FBP, is positively regulated by fructose 2,6-bisphosphate. And the production of fructose 2,6-bisphosphate is something that can be inhibited by ATP, inhibited by phosphoenolpyruvate, and activated by ATP. So exactly what is shown here is allosteric regulation. Just some of this allosteric regulation is via this side enzyme to make a separate product, fructose 2,6-bisphosphate, and there's control theory considerations for why that might happen. All right. So at this point, we've learned how we can start with glucose, oxidase that glucose via a pathway that now allows us to get the energy that cells need to maintain ATP-ADP ratio in a good physiological range despite the high ATP-ADP ratio in cells. However, it should also be clear that in order for this to work, we need a way to actually get that glucose from somewhere to begin with. You probably learned in high school that plants use photosynthesis as a way to get energy from the sun. Well, the synthesis part of photosynthesis is generating the sugar that those plants can then use to burn and keep ATP-ADP high at night and survive the night until the sun comes up again when they can again use energy from the sun directly to get energy. We as animals evolve the way that we have to eat the plants. We have to get the glucose that the plants make, but of course, we also use-- generate-- have the ability to generate some glucose, and this is best illustrated from muscle physiology. And that is, we alluded to earlier, that if we're running away from the lion, our muscles might outstrip the supply of oxygen in the blood and basically generate a whole lot of lactate. Well, that lactate is perfectly good fuel. Just like the yeast, can oxidize or we can reoxidize ethanol. We can also use lactate as a fuel, but it turns out, a big job of our liver is to take that lactate from the blood, as well as other molecules, and regenerate glucose so that our blood glucose remains in a constant level. This cycling of glucose and lactate across the body between the muscle and the liver is sometimes referred to as the Cori cycle and involves a clear set of reactions to turn that lactate back into glucose, a process called gluconeogenesis. And unfortunately we're out of time today, but next time I will start off the lecture by going through how it is that gluconeogenesis works to turn something like lactate back into glucose so that organisms have the glucose to begin with to run glycolysis. Thank you.

★本章小结：核心代谢逻辑链

1. 一条能量主线（为什么生命一刻不停地做分解代谢？）：驱动吸能反应的是高 [ATP]/[ADP] 比值（及 [ATP]/[AMP] 能荷）。短期应急靠 磷酸肌酸/肌酸激酶系统 和 腺苷酸激酶（2ADP ⇌ ATP + AMP） 缓冲；持续充电靠分解还原态碳源（葡萄糖 → 丙酮酸 → CO₂）。
2. 十步反应两阶段（细胞质/胞液，有氧无氧皆可）：

• 准备期（1–5 步，耗 2 ATP）：Glc ──(① HK/GK (-ATP))──► G-6-P ⇌(② PGI) F-6-P ──(③ PFK-1 (-ATP))──► F-1,6-BP ⇌(④ 醛缩酶) DHAP + G3P ⇌(⑤ TPI) 2 G3P。
• 获能期（6–10 步，产 4 ATP + 2 NADH，全部 × 2）：2 G3P ⇌(⑥ GAPDH（唯一氧化步）｜+2 P_i, +2 NADH) 2 1,3-BPG ⇌(⑦ PGK (+2 ATP，净ATP归零)｜第一次底物水平磷酸化) 2 3-PG ⇌(⑧ PGAM｜2,3-BPG 引物) 2 2-PG ⇌(⑨ 烯醇化酶 (-2 H_2O)｜F^-抑制) 2 PEP ──(⑩ PK (+2 ATP)｜第二次底物水平磷酸化)──► 2 丙酮酸。

1. 两个核心有机化学机制：① GAPDH 利用活性中心 Cys-SH 进攻醛基成硫半缩醛 → 把 H^- 交给 NAD⁺ 生成 高能酰基-酶硫酯中间物 → 无机磷酸 P_i 磷酸解生成高能酰基磷酸 1,3-BPG（碘乙酸锁死 -SH；砷酸盐 AsO₄^3- 替代 P_i 解偶联使净产 ATP=0）。② PK 催化的高能推力源于 烯醇式丙酮酸自发互变异构为酮式丙酮酸（Δ G'^°_PEP水解 = -61.9 kJ/mol）。
2. 三条丙酮酸去路与电子收支守恒：无氧时为使 NADH → NAD⁺ 再生以维持 GAPDH 运转，动物肌肉/红细胞经 LDH 将丙酮酸还原为 L-乳酸（净产 2 ATP）；酵母经 丙酮酸脱羧酶（TPP 碳负离子电子阱，放 CO₂） 和 乙醇脱氢酶（Zn²⁺） 还原为 乙醇（净产 2 ATP）；有氧时胞质 2 NADH 经穿梭入线粒体（酵解阶段合 5 或 7 ATP），丙酮酸进线粒体经 PDH 和 TCA 彻底氧化（全过程 6 次底物水平磷酸化，共得 30 或 32 ATP，旧口径 36 或 38 ATP）。
3. 三大不可逆限速酶与肿瘤代谢重编程：HK/GK（入口锁糖）、PFK-1（真正承诺步，受 F-2,6-BP 最强激活，受双功能酶 PFK-2/FBPase-2 磷酸化共价调控，受 ATP 和柠檬酸抑制、AMP 激活）、PK（出口阀，受 F-1,6-BP 前馈激活，肝 L-PK 受 PKA 磷酸化失活）；肿瘤细胞通过 Warburg 效应 与低活性二聚体 PKM2 将糖酵解中间产物分流向核糖、丝氨酸与脂质合成，满足增殖生物量需求。

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★名词解释速背卡（中英对照高频名解）

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★易混概念与新旧教材口径辨析

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★华农历年真题章末综合自测

在第二章中，我们跟随 MIT 7.05 的 Matthew Vander Heiden 教授走完了糖酵解（EMP）的十步拆解之旅：1 分子六碳葡萄糖在胞质中裂解为 2 分子三碳丙酮酸，净赚 2 分子 ATP 和 2 分子 NADH。但在第 22 讲和第 23 讲的课堂黑板前，Vander Heiden 教授抛出了一连串更贴近生命全貌的追问：第一，当你剧烈冲刺跑完 800 米、肌肉排出大量乳酸，或者当你深夜空腹入睡、长达十几个小时不进食时，你的大脑和红细胞每秒钟都在嗷嗷待哺地消耗葡萄糖——既然“所有酶促反应在微观上都可逆”，为什么肝脏不能干脆把糖酵解倒着开回去，直接用 2 分子丙酮酸和 2 分子 ATP 逆行合成葡萄糖？第二，当你饱餐一顿碳水化合物后，身体如何用极低的能量代价把多余的葡萄糖打包成高度分支的糖原颗粒？为什么肾上腺素触发的一个磷酸化信号，竟能同时开启糖原分解的大门，又反手把糖原合成的大门焊死？第三，当氧气充足时，丙酮酸没有去发酵成乳酸，而是跨过线粒体内膜，被一台由“三酶五辅酶”精密组装的纳米机器——丙酮酸脱氢酶复合体（PDH）转化为乙酰-CoA，进而汇入 Hans Krebs 发现的柠檬酸循环（TCA）；在这个看似对称的循环里，为什么给细胞喂下带有 ¹⁴C 放射性标记的乙酰-CoA，第一轮循环脱下的两分子 CO₂ 竟然连一丝一毫放射性都没有？为什么春天萌发的向日葵种子能把满肚子的油脂变成甜甜的糖，而吃下肥肉的人类却永远无法把偶数碳脂肪酸变成一分子葡萄糖？

这一连串问题，直指细胞碳骨架与能量调度的四大中枢机制：糖异生（Gluconeogenesis）、糖原合成与分解（Glycogen Metabolism）、丙酮酸脱氢酶复合体（PDH Complex）以及柠檬酸循环与乙醛酸循环（TCA & Glyoxylate Cycles）。它们共同构成了以丙酮酸、草酰乙酸、6-磷酸葡萄糖和乙酰-CoA 为四大十字路口的立体代谢网络。无论是在 MIT 7.05 的考卷上，还是在华中农业大学生物化学考研（338/802）的历年真题中，本章都是当之无愧的“压轴大题发动机”——能量大账单（30/32 ATP 计算）、同位素追踪、激素级联放大与三大物质代谢共同通路，几乎年年必考。现在，就让我们走进 MIT 7.05 L22–L23 的课堂，把这四大模块从热力学与有机化学底层逻辑一举打通！

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3.1糖异生（Gluconeogenesis）：为什么不能直接逆转糖酵解？三步能障的精妙绕道与跨膜协同

一、热力学死胡同：为什么不能把糖酵解直接倒着开？

在 L22 开场时，Vander Heiden 教授指着黑板上的糖酵解自由能阶梯图提出了第一个核心问题：当我们处于饥饿状态或剧烈运动后，肝脏需要把乳酸、丙酮酸等三碳分子重新拼成六碳葡萄糖来维持血糖稳定。既然我们在酶学中学过“酶只降低活化能、不改变平衡常数，原则上每一步酶促反应都可逆”，那为什么细胞不直接用糖酵解的那 10 种酶，原路退回合成葡萄糖呢？

让我们算一笔热力学账。在糖酵解的 10 步反应中，有 7 步反应的生理自由能变 Δ G 接近于 0（处于准平衡态），只要改变底物与产物的浓度比，同一个酶确实可以顺畅地双向催化。然而，己糖激酶（HK）、磷酸果糖激酶-1（PFK-1）和丙酮酸激酶（PK）催化的三步反应 2013/2014/2016·判断 具有巨大的负自由能变（Δ G ≪ 0），犹如糖酵解河道上的三级陡峭瀑布。如果强行把糖酵解直接逆转（只消耗 2 分子 ATP），总反应的 Δ G'^° 高达 +75～ +84 kJ/mol（约 +20 kcal/mol），平衡常数极小，在生理条件下根本不可能逆向进行！

因此，生物进化采取了极其聪明的“借道超车”策略：糖异生（Gluconeogenesis）：是指以非糖物质（如乳酸、丙酮酸、甘油、生糖氨基酸、丙酸等）为前体合成葡萄糖（或糖原）的过程 2011/2023/2025·名解。它与糖酵解绝非简单逆转，而是共用 7 步近平衡可逆反应，并用 4 种特有的关键酶绕过糖酵解的 3 步不可逆能障，通过额外投入高能磷酸键把总反应的 Δ G'^° 扭转为 -37.7 kJ/mol（约 -9 kcal/mol），使葡萄糖的净合成在热力学上完全自发可行！

二、三大能障的绕道化学机制：为什么丙酮酸变 PEP 要“先加 CO₂ 再脱 CO₂”？

为什么绕过 3 步不可逆反应却需要 4 种酶？因为后两步能障（F-1,6-BP → F-6-P 和 G-6-P → Glc）只需用磷酸酶直接水解掉磷酸酯键即可放能过关；唯独第一步能障——丙酮酸 → 磷酸烯醇式丙酮酸（PEP），面临着极其严峻的化学挑战。

我们在第二章学过，PEP 的高能烯醇磷酸键水解自由能高达 Δ G'^° = -61.9 kJ/mol（主要由烯醇式丙酮酸自发互变异构为稳定的酮式丙酮酸所驱动）。哪怕细胞内 ATP/ADP 比值再高，单凭 1 分子 ATP 水解（Δ G'^° = -30.5 kJ/mol）的能量，也绝对不足以把稳定的酮式丙酮酸硬推回高能的烯醇磷酸酯 PEP！为了翻越这座高达 61.9 kJ/mol 的能量高山，细胞设计了一套精妙绝伦的“先羧化激活、后脱羧磷酸化”两步联用策略，分摊由 1 分子 ATP 和 1 分子 GTP 共同买单（即“先 A 后 G”）：

1. 第一关绕道（两步酶促 + 跨膜穿梭）：丙酮酸 → 草酰乙酸 → PEP

• 第一步：丙酮酸羧化酶（Pyruvate Carboxylase, PC）催化的生物素羧化反应（线粒体基质）
  丙酮酸 + HCO₃^- + ATP ──(丙酮酸羧化酶（生物素、Mg²⁺、乙酰-CoA 激活）)──► 草酰乙酸（OAA） + ADP + P_i + H⁺
  Vander Heiden 教授在黑板上详细画出了这步反应的辅基机制：丙酮酸羧化酶是一个仅存在于线粒体基质中的四聚体酶，以生物素（Biotin，维生素 B₇）为共价辅基 2008·单选2025·判断。生物素的戊酸侧链羧基与酶活性中心特定赖氨酸（Lys）残基的 ε-氨基形成酰胺键，构成一条长约 1.4 nm 的柔性摆臂（生物素赖氨酰基，biocytin）。催化分为两个活性亚位点进行：

1. 碳酸氢盐磷酸化与生物素羧化（亚位点 1）：酶并不直接使用游离 CO₂，而是利用水溶性的 HCO₃^-。ATP 先将 HCO₃^- 磷酸化生成高活性的羧基磷酸（carboxyphosphate）中间体，随即释放 P_i，将活化的羧基转移到生物素脲环的 N¹ 位上，形成高能的 N¹-羧基生物素（N¹-carboxybiotin）。
2. 摆臂转移与丙酮酸亲核进攻（亚位点 2）：共价长臂将 N¹-羧基生物素摆动到第 2 个催化位点；在此处生物素脱羧释放出高活性的 CO₂，同时丙酮酸甲基去质子化形成烯醇负离子（enolate），亲核进攻该 CO₂，形成新的碳-碳键，生成四碳的草酰乙酸（OAA）！

• 为什么丙酮酸羧化酶要专门放在线粒体基质中？ Vander Heiden 教授从区室化（Compartmentalization）角度给出了漂亮解释：第一，线粒体是氧化磷酸化的场所，基质内維持着极高的 [ATP]/[ADP] 比值，能强力驱动耗能的羧化反应；第二，线粒体基质因呼吸链不断向外泵出 H⁺ 而呈偏碱性（pH ≈ 7.8），高 pH 有利于溶解的 CO₂ 转化为底物 HCO₃^-！
• 丙酮酸羧化酶的绝对别构激活剂是乙酰-CoA（Acetyl-CoA） 2008·单选：若没有乙酰-CoA 结合，生物素根本无法发生羧化！这背后有着深刻的生理智慧：当线粒体内乙酰-CoA 充足时，要么说明能量过剩（来自脂肪酸 β-氧化），此时激活 PC 可将丙酮酸推向糖异生；要么说明 TCA 循环因缺乏载体草酰乙酸而拥堵，此时激活 PC 可生成草酰乙酸执行回补反应（Anaplerosis），接纳乙酰-CoA 进入 TCA 循环彻底氧化！

• 跨膜难题与“一石二鸟”的苹果酸穿梭：草酰乙酸如何出线粒体？
  糖异生后续的反应都在细胞质基质中进行，但线粒体基质生成的草酰乙酸却遇到了一个尴尬的障碍：线粒体内膜上根本没有草酰乙酸的转运蛋白，草酰乙酸无法直接跨膜进入胞质！细胞如何破解这一困局？

1. 以丙酮酸（或生糖氨基酸）为原料时的“苹果酸路线”：线粒体基质中的草酰乙酸先由线粒体苹果酸脱氢酶（mMDH）消耗 1 分子线粒体 NADH，逆向还原为L-苹果酸（Malate）；苹果酸通过线粒体内膜上的苹果酸-α-酮戊二酸转运载体堂而皇之地跨膜进入细胞质；到了胞质后，再由胞质苹果酸脱氢酶（cMDH）将其重新氧化为草酰乙酸，同时在胞质中生成 1 分子胞质 NADH！
   为什么草酰乙酸要大费周章先变成苹果酸出线粒体，到了胞质再变回草酰乙酸？ 因为这实现了“一石二鸟”：第一，给没有通行证的草酰乙酸换上“苹果酸伪装”跨过线粒体内膜；第二，糖异生在胞质进行到 1,3-二磷酸甘油酸（1,3-BPG） → 3-磷酸甘油醛（GAP）（GAPDH 逆反应）时，急需消耗胞质 NADH，而正常有氧条件下胞质 [NADH]/[NAD⁺] 比值极低（约 10^-5～ 10^-4）！苹果酸跨膜路线恰好把线粒体丰富的 NADH 等当量地“搬运”到了胞质，完美解决了糖异生的还原力需求！
2. 以乳酸为原料时的“PEP 直接出膜路线”：如果糖异生的起点是骨骼肌送来的乳酸，情况就不同了！乳酸在肝细胞质中被乳酸脱氢酶（LDH）氧化为丙酮酸时（乳酸 + NAD⁺ → 丙酮酸 + NADH + H⁺），已经在细胞质中净生成了 1 分子 NADH，正好够后面的 GAPDH 逆反应使用，不再需要从线粒体搬运 NADH！因此，丙酮酸进入线粒体羧化成草酰乙酸后，可直接由线粒体型 PEPCK 在基质内转化为 PEP，再经内膜上的三羧酸/PEP 载体直接转运出线粒体（也可经天冬氨酸转运出膜）。

• 第二步：磷酸烯醇式丙酮酸羧激酶（PEPCK）催化的脱羧磷酸化反应
  草酰乙酸（OAA） + GTP ──(PEPCK（Mg²⁺/Mn²⁺）)──► 磷酸烯醇式丙酮酸（PEP） + GDP + CO₂↑
  请仔细观察草酰乙酸的结构（^-OOC-CO-CH_2-COO^-）：它的酮羰基相对于一端的羧基是 α-位，而相对于另一端（刚刚由丙酮酸羧化酶接上去的那个羧基！）则是 β-位。也就是说，草酰乙酸既是一个 α-酮酸，更是一个不稳定的 β-酮酸！在有机化学中，β-酮酸极易发生自发脱羧释放出巨大自由能，并在脱羧瞬间于 C2–C3 间形成共振稳定的烯醇负离子（enolate）中间体；此时该烯醇氧原子立即亲核进攻 GTP 的 γ-磷原子，将丙酮酸牢牢“捕获（trap）”在高能的烯醇磷酸酯（PEP）状态！
• 同位素标记大坑：在丙酮酸羧化酶（PC）步骤接上去的那一分子 HCO₃^-（CO₂），在紧接着的 PEPCK 步骤中被原封不动地脱掉了！ 也就是说，CO₂ 在这里扮演了“催化剂式能量搬运工”：先耗 1 分子 ATP 把 CO₂ 接上去把丙酮酸改造成 β-酮酸（草酰乙酸），随后利用脱去同一个 CO₂ 释放的化学能，协助 GTP 完成高难度的烯醇磷酸化（总 Δ G'^° = +0.8 kJ/mol，生理条件下完全倾向于生成 PEP）！

2. 第二关绕道：果糖-1,6-二磷酸酶-1（FBPase-1，细胞质基质）

生成 PEP 后，碳骨架沿着糖酵解的 7 步可逆反应一路顺畅逆行（PEP → 2-PG → 3-PG ──(-ATP)──► 1,3-BPG ──(-NADH)──► GAP ↔ DHAP → F-1,6-BP），直到撞上第二座能量高墙——磷酸果糖激酶-1（PFK-1）。为了绕过它，细胞动用糖异生最关键的限速酶：果糖-1,6-二磷酸酶-1（FBPase-1，又称 1,6-二磷酸果糖磷酸酶）：

果糖-1,6-二磷酸（F-1,6-BP） + H_2O ──(FBPase-1（Mg²⁺）)──► 6-磷酸果糖（F-6-P） + P_i (Δ G'^° = -16.3 kJ/mol)

注意：这步反应不是把磷酸基转给 ADP 生成 ATP，而是由磷酸酯酶催化 C1 位磷酸酯键的不可逆水解，释放出游离无机磷酸 P_i，从而获得显著的负自由能变推动反应前行。

3. 第三关绕道：葡萄糖-6-磷酸酶（G-6-Pase，光面内质网腔）

F-6-P 经磷酸己糖异构酶可逆转化为 6-磷酸葡萄糖（G-6-P）后，来到了最后一道关卡——绕过己糖激酶/葡糖激酶。催化这步反应的酶是 葡萄糖-6-磷酸酶（G-6-Pase，又称 6-磷酸葡糖磷酸酶）：

6-磷酸葡萄糖（G-6-P） + H_2O ──(葡萄糖-6-磷酸酶（光面内质网）)──► 葡萄糖（Glucose） + P_i (Δ G'^° = -13.8 kJ/mol)

三、糖异生的能量总账、前体资格审查与双器官协作循环

1. 糖异生能量细账：合成 1 分子葡萄糖为什么要耗 6 个高能磷酸键？

让我们跟随 Vander Heiden 教授把从 2 分子丙酮酸合成 1 分子葡萄糖的能量账单逐笔算清（因为每个葡萄糖有 6 个碳，而丙酮酸只有 3 个碳，所有三碳步骤必须 × 2）：

1. 丙酮酸 → 草酰乙酸（丙酮酸羧化酶 × 2）：消耗 2 ATP；
2. 草酰乙酸 → PEP（PEPCK × 2）：消耗 2 GTP（经核苷二磷酸激酶 ATP + GDP ↔ ADP + GTP，Δ G'^° ≈ 0，能量等价于 2 ATP）；
3. 3-磷酸甘油酸（3-PG） → 1,3-二磷酸甘油酸（1,3-BPG）（磷酸甘油酸激酶 PGK 逆向反应 × 2）：消耗 2 ATP；
4. 1,3-二磷酸甘油酸（1,3-BPG） → 3-磷酸甘油醛（GAP）（3-磷酸甘油醛脱氢酶 GAPDH 逆向反应 × 2）：消耗 2 NADH + 2 H⁺（胞质还原力）；
5. FBPase-1 和 G-6-Pase 两步水解反应：释放 2 P_i，不消耗也不产生 ATP。

因此，糖异生总反应式为：2 丙酮酸 + 4 ATP + 2 GTP + 2 NADH + 2 H⁺ + 6 H_2O → 葡萄糖 + 4 ADP + 2 GDP + 6 P_i + 2 NAD⁺ 2011/2018·简答。

也就是说，从 2 分子丙酮酸合成 1 分子葡萄糖，总共需要消耗 4 ATP + 2 GTP = 6 个高能磷酸键（外加 2 分子 NADH；这些 ATP 和 NADH 在饥饿期主要由肝细胞线粒体中的脂肪酸 β-氧化提供）！相比之下，1 分子葡萄糖经糖酵解裂解为 2 分子丙酮酸只净赚 2 分子 ATP。多付出的这 4 个高能磷酸键（2 ATP + 2 GTP），正是为了克服热力学壁垒、让糖异生自发向前推进所必须支付的“热力学过路费”！

2. 糖异生前体的“资格审查”：谁能变糖，谁绝不能变糖？

并非所有非糖物质都能在动物体内变成葡萄糖，这取决于它能否净生成草酰乙酸（OAA）或磷酸丙糖（DHAP/GAP）：

1. 乳酸与丙酮酸：最核心的三碳前体，经 PC + PEPCK 途径耗 6 个高能键合成葡萄糖。
2. 甘油（Glycerol）：脂肪动员（甘油三酯水解）释放的甘油不能在脂肪细胞原地利用（脂肪细胞缺乏甘油激酶 2024·单选），必须经血液运到肝脏或肾脏。在肝细胞质中，甘油先由甘油激酶耗 1 分子 ATP 磷酸化为 3-磷酸甘油（α-磷酸甘油），再由胞质 3-磷酸甘油脱氢酶（NAD⁺ 型）氧化为磷酸二羟丙酮（DHAP）（同时生成 1 分子胞质 NADH）。注意：甘油从 DHAP 直接汇入糖异生中段，不经过草酰乙酸，因此只需绕过 FBPase-1 和 G-6-Pase 两步不可逆反应！由 2 分子甘油合成 1 分子葡萄糖仅需消耗 2 ATP，且净产生 2 NADH。
3. 生糖氨基酸与三羧酸循环中间物：体内 20 种标准氨基酸中，除 亮氨酸（Leu）和赖氨酸（Lys）是纯生酮氨基酸外，其余 18 种氨基酸均可直接转化为丙酮酸或 TCA 循环中间物（α-酮戊二酸、琥珀酰-CoA、延胡索酸、草酰乙酸），进而净生成草酰乙酸进入糖异生。
4. 奇数碳脂肪酸与丙酸：奇数碳脂肪酸 β-氧化最后一轮产生 1 分子三碳的丙酰-CoA（反刍动物瘤胃发酵也产生大量丙酸），经羧化和变位酶（需辅酶 B₁₂）转化为四碳的琥珀酰-CoA，可净增草酰乙酸而进入糖异生。
5. 绝对禁区：在动物体内，乙酰-CoA（以及偶数碳脂肪酸、乙醇）绝对不能作为糖异生的原料净合成葡萄糖 2012/2026·判断！原因有二：① 丙酮酸脱氢酶复合体（PDH）催化的 丙酮酸 → 乙酰-CoA + CO₂ 反应高度放能，绝对不可逆；② 乙酰-CoA（2C）进入 TCA 循环后，虽然与草酰乙酸（4C）缩合成柠檬酸（6C），但在一轮循环中必经两次氧化脱羧释放 2 分子 CO₂，循环结束时草酰乙酸没有丝毫净增加（净增碳数 = 2 - 2 = 0），且动物体内缺乏植物/微生物特有的乙醛酸循环酶系！

3. 器官间代谢接力：Cori 循环（乳酸循环）与葡萄糖-丙氨酸循环

在人体内，骨骼肌与肝脏之间通过血液建立起了两条著名的碳/氮跨器官接力通道（均是华农真题三年一考的五星级名解！）：

• 乳酸循环（Cori Cycle）：是指剧烈运动时骨骼肌（或无氧酵解的红细胞）分解葡萄糖产生的乳酸，经血液运输到肝脏，在肝内经糖异生途径重新合成葡萄糖，再随血液运回骨骼肌和脑组织被利用的跨器官循环过程 2007/2016/2025·名解。
• 生理意义：① 防止乳酸酸中毒（骨骼肌缺乏 G-6-Pase，且乳酸是代谢的“死胡同”，必须转回丙酮酸才能代谢）；② 回收乳酸中的碳骨架与化学能，将肌肉的代谢负担转移给氧气充足、富含线粒体的肝脏；③ 维持血糖恒定并更新肝糖原。
• 能量账单：在骨骼肌中，1 分子葡萄糖无氧酵解生成 2 分子乳酸，净赚 2 ATP；在肝脏中，2 分子乳酸异生回 1 分子葡萄糖，消耗 4 ATP + 2 GTP = 6 个高能磷酸键（LDH 逆反应生成的 2 NADH 正好抵消 GAPDH 逆反应所需的 2 NADH）。因此，每运转一轮 Cori 循环，机体净消耗 6 - 2 = 4 个高能磷酸键！
• 葡萄糖-丙氨酸循环（Glucose-Alanine Cycle）：是指骨骼肌中的氨基酸经转氨基作用将氨基交给丙酮酸生成丙氨酸（由谷丙转氨酶 ALT/GPT 催化），丙氨酸呈电中性进入血液运至肝脏，在肝内再次转氨还原为丙酮酸用于糖异生合成葡萄糖运回肌肉，而脱下的氨基则在肝内进入尿素循环合成尿素排出的过程 2010/2013/2016/2018·名解。
• 与 Cori 循环的核心对比：两者都是“肌肉送三碳糖异生前体去肝、肝送六碳葡萄糖回肌肉”；但 Cori 循环只运碳不运氮，而葡萄糖-丙氨酸循环依赖谷丙转氨酶（ALT，绝非谷草转氨酶 AST！），既为肝脏提供糖异生碳骨架，又以无毒的丙氨酸形式将肌肉产生的氨安全转运至肝脏解毒！此外，丙氨酸进入肝细胞后还能别构抑制肝丙酮酸激酶，确保转氨生成的丙酮酸全力流向糖异生。

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🎧 MIT 7.05 课堂原声英文实录对照（本节关联 1 讲 · 完整逐段实录）点击展开 / 收起 ▾

🎬 MIT 7.05 Lec 22 · Gluconeogenesis, Glycogen & TCA Cycle I⏱ ~50:00

~00:00MITOCW | 22. Glucogenesis/Carbohydrate Storage/TCA Cycle I

~00:02PROFESSOR:

~00:02So last time, we discussed glycolysis as a pathway. And I just want to remind everybody that, like all pathways, glycolysis has to be favorable. That is, delta G across the entire pathway has to be less than 0. We spent a lot of time discussing the regulation of glycolysis. And I just want to revisit this quickly. And remember, we discussed that the steps that are regulated are those with the biggest, quote, unquote, bio-energetic cost. That is, the biggest drop in delta G is as molecules would proceed across the pathway. And those are shown here-- the hexokinase step, the phosphofructokinase step, and the pyruvate kinase step of the pathway. Now this is largely true of most pathways that we will consider, in that these regulated steps are often those with the biggest bio-energetic cost. And the reason for that is that it makes a lot of sense. Once you go through those steps, it's very difficult to go back.

~00:55Also, you'll note that these are the steps that regulate entrance and exit from the pathway. And this also is something we'll commonly see and other pathways and also makes some sense. Just to remind everybody, I drew up here a skeletonized version of glycolysis and the most important regulatory features of it. Just to remind people that initially, hexokinase is regulated by glucose-6 phosphate. Glucose-6 phosphate negatively regulates hexokinase. And that's because if the glucose-6 phosphate levels in the cell go up such that it can't be shunted to glycogen-- something we'll talk about today-- or be sent through glycolysis, it makes sense not to trap more glucose. Once glucose-6 phosphate enters the rest of the glycolytic pathway, the key step of regulation is phosphofructokinase. It, of course, will have positive feedback, saying, let's put carbon into the pathway. If energy charge low, AMP is high, activates it. This molecule fructose 2,6-bisphosphate that we talked about earlier today-- we'll mention again today-- can be involved in some other signaling inputs to allosterically regulate the enzyme. FBP can feed forward to activate pyruvate kinase. You want to match input with output. And, of course, if PEP builds up, you want to slow it down. If a downstream product-- citrate-- builds up, you want to slow it down. And of course, a major output of glycolysis is ATP. And if ATP is high, no reason to continue oxidizing glucose carbons. Now this regulation we discussed is often done at the allosteric level by controlling enzyme rates. That can be via a small molecule binding, as I've shown up here. And it could also be via signaling. And that's really a place where fructose 2,6-bisphosphate as a combination of signaling, and allosteric small molecules comes into play. That is, you can have signaling that controls the enzymes that generate fructose 2,6-bisphosphate, thereby creating an additional molecule that can act as allosteric regulator of the enzyme PFK to generate FBP and commit carbon to glycolysis.

~02:44Now we closed the discussion last time beginning to discuss how it is that organisms can make glucose. Obviously, this glucose carbon that is being oxidized to get ATP has to come from somewhere. We mentioned that plants make glucose during the day when they have plenty of excess energy from light from the sun but then use that glucose to do constant metabolism to maintain their ATP levels at night. And we as animals obviously take advantage of this by eating the glucose that ultimately was created by plants using the energy of the sun. We began to discuss an example of where us, as organisms, also make glucose. Of course, this is not net production of glucose. But for instance, if our muscle cells are working hard, lactate can build up. That lactate is excreted into the blood. That lactate can then be used by the liver to regenerate glucose. And one of the major jobs of the liver is to maintain a constant level of blood glucose to continue to supply fuel for various organs, including the muscle to be able to carry out the work that it needs to do. And so this whole process is called the Cori cycle. And a major job of the liver is thus generating glucose to maintain blood glucose levels. And the pathways used to do this is a pathway that we began to talk about last time called gluconeogenesis. And it's really a discussion of gluconeogenesis. That is, how can one build a pathway in plants, in the liver, wherever, as a way to generate glucose from something like lactate? Now, of course, we know from last time, lactate dehydrogenase can, obviously, it's a redox reaction to interconvert with pyruvate. And what gluconeogenesis really is it's a pathway that cells use to convert pyruvate or really anything else that can come into glycolysis-- and example being lactate. This, of course, can happen by the lactate dehydrogenase reaction. And then once it has that pyruvate, turn that pyruvate into glucose. That's really what we're talking about when we're discussing gluconeogenesis. Now how can we turn pyruvate into glucose? Well, we just discussed the whole pathway where we could turn glucose into pyruvate-- glycolysis. And so this is really the reverse of glycolysis. I said many times that all enzymes are reversible. So why can't we just run the pathway in the opposite direction? Well, hopefully, if you've been paying attention for the last several lectures, you realize there's two problems with doing that. The first is that, remember, why we're doing metabolism in the first place is to maintain a high ATP:ADP ratio under conditions so that the cell can use that ATP:ADP ratio to carry out otherwise unfavorable processes. And so thus, at a very high level, cells can't stop catabolism. Glycolysis or whatever has to continue to maintain ATP in the right range. And so now, if we're going to do something anabolic-- that is, build something, turn it into glucose, this has to happen in the background where catabolism has still happened. And so at least you need some kind of way to control both and prevent this from being some kind of futile cycle. Second, even if that ATP level is super high in the cell, it has extra energy, there is still a thermodynamic problem with just reversing glycolysis. And that is, remember, delta G has to be less than 0 for any pathway to work. And so it then stands to reason that if glucose oxidation to pyruvate, if this reaction is favored, as it is in glycolysis, that delta G is less than 0.

~06:03Well, under the conditions where that's occurring, if we want to then say, well, what about the reverse? Let's switch the signs-- pyruvate to glucose. We know that delta G is the same as it was in the opposite direction but, now, changing the signs from positive to negative or negative positive. And so, thus, delta G, for the reverse reaction, must be greater than 0 and, therefore, must be spontaneous-- is not spontaneous. And therefore says that we need to have a different pathway simply for energetic reasons if we're going to go in the opposite direction. Now oxidizing glucose releases energy. That's how we keep the ATP:ADP ratio high. It stands to reason then that we are going to have to put energy in if we're going to run in the opposite direction. And so if we're going to build a pathway from pyruvate to glucose, you can guess, we're going to have to add couple that pathway to ATP-ADP hydrolysis-- that is, couple it to something favorable to now allow this otherwise unfavorable reaction to happen. Now this energy, of course, in plants can come from the sun. We'll discuss that in great detail when we get to photosynthesis. However, for the sake of you and I, I guess, what it says is, if you think about this, if you release energy from glycolysis, it makes sense then you have to add energy if you're going to do gluconeogenesis. But remember, to satisfy all the laws of thermodynamics, the net energy loss to the universe is going to be-- must exist, which means that it is going to cost more energy to make glucose than you're going to get back from breaking it down. That just, hopefully, fits with your intuition. And it actually, I guess, is good news, because when are we going to do anabolism? When are we going to store all these excess calories as glucose? Well, we're going to do it if we overeat. And if we overeat, and it costs us energy in the form of calories to actually store that energy for later, I guess, it means that when we overeat, we don't have things quite as bad as it could have been. All right. Now let's get back to the how one can actually do this. So if we want to think, what is a pathway that we can build to get from pyruvate back to glucose? Well, we know we're going to have to burn some ATP to do it. But what are the issues with simply reversing glycolysis? Well, we can come back here and say, well, what was driving glycolysis in the first place? And it was these big steps of free energy change acts of hexokinase, phosphofructokinase, and pyruvate kinase. Where as many of these other reactions, at least under these standard conditions that are estimated to exist in cells, are relatively close to equilibrium. So there's really no problem with reversing glycolysis across here or across there. The real issue comes from getting around the hexokinase to phosphofructokinase and the pyruvate kinase step. These are the places where the most energy is released, if you will, when you're going through glycolysis. So those are the steps we're going to have to add energy in order to reverse build a pathway that goes in the opposite direction and allows you to start with pyruvate and end up with glucose. And so what we will find is that gluconeogenesis and glycolysis use many of the same enzymes. However, not all of the enzymes are the same because we need different enzymes if we're going to get around this hexokinase, phosphofructokinase, and pyruvate kinase steps of the pathway. And so for gluconeogenesis, we essentially need four new enzymes in order to get past those places. So we're going to need a new enzyme called glucose 6phosphatase. And glucose 6-phosphatase is going to allow us to get from glucose 6-phosphate and turn it back into glucose.

~09:41All right. We're going to need an enzyme fructose 1,6-bisphosphatase, which is going to allow us to take FBP and turn it back into fructose 6-phosphate. And then we're going to need two enzymes to get around the pyruvate kinase step. These are two enzymes called PEPCK and PC. That stands for phosphoenolpyruvate carboxykinase and pyruvate carboxylase. I'll discuss these in great detail in a minute. And these are going to allow us to get from pyruvate back to PEP. Also, essentially, I'll add them up here to this diagram. So to get back from pyruvate to PEP, two enzymes-pyruvate carboxylase and phosphoenolpyruvate carboxykinase to get from fructose 1,6-bisphosphate, fructose 6phosphate, an enzyme called FBPase. And to get from glucose 6-phosphate back to glucose, an enzyme called glucose 6-phosphatase. OK. Now, of course, if you think back, we're also going to need to balance electrons in the pathway. Remember, glucose to pyruvate is an oxidation reaction. That is, we're generating NADH. That means to go in the reverse direction, we need a source of NADH to reverse that GAPDH reaction. I just want to point out that you're only doing gluconeogenesis if cells have plenty of energy. What does plenty of energy mean? Well, that means the ATP:ADP ratio is high because you're doing lots of metabolism. But if you're doing lots of metabolism, and the ATP:ADP ratio's high, that also means the NADH/NAD+ ratio is high. And so you can think of the NAD regeneration by gluconeogenesis as being, for all intents and purposes, an alternative to fermentation, if you will, as a way to keep catabolism going. That is, you're only doing this if you have plenty of NADH around. And so dealing with that electron balance is less of an issue. OK. Now let's discuss briefly how some of these enzymes work. So glucose 6-phosphate and fructose 1,6bisphosphatase are very straightforward. Effectively, all they are is simply removing a phosphate. That's very favorable. Remember, when we did those reactions of hexokinase phosphofructokinase reactions and glycolysis, that, we had to couple otherwise unfavorable things-- adding a phosphate group to ATP hydrolysis. This is the reverse of that. And so the reverse of that, delta G is going to be less than 0. Delta G less than 0 means we just have a phosphatase there, just like you might have on a kinase enzyme. That's very straightforward. But there's a much bigger issue to get around this other step, this pyruvate kinase step. Remember, equilibrium strongly favored PEP to pyruvate conversion. That was one of the reasons why we could use that step to maintain a high ATP:ADP ratio in cells. Well, now, the issue becomes, how can we trap that phosphate back onto pyruvate while generating phosphoenolpyruvate? How one does that is an issue. Even if the ATP:ADP ratio is high, it's never high enough to reverse pyruvate kinase on its own. And so we need a new pathway with two steps in it. That's why there's two enzymes-- PC and PEPCK-- that are there to create a pathway where pyruvate to PEP now becomes favorable. That is, where delta G 0 prime is less than 0. That is, the equilibrium favors to the right. And under cellular conditions, this pathway can actually happen such that cells can net make glucose. Now these are two steps, both of which require ATP. And so you effectively get one ATP from PEP to pyruvate conversion. It costs you to ADPs or two ADP equivalents to go back from pyruvate to PEP, which makes sense. You should have to spend more energy to go upstream than you get back coming downstream. All right. Here's how this pathway works.

~13:03So this is pyruvate, the first reaction catalyzed by PC, which stands for pyruvate carboxylase. Couples ATP hydrolysis to addition of a CO2 that generates this intermediate, which is called oxaloacetate, often abbreviated OAA for Oxaloacetic Acid. That oxaloacetate is then decarboxylated. That is, the CO2 that is lost is removed-- that was added is removed. This reaction is coupled to GTP and is catalyzed by this enzyme, PEPCK PEP carboxykinase and uses that phosphate from GTP to trap pyruvate in the enol form as PEP or phosphoenolpyruvate. great. So once you've regenerated PEP, now that PEP gets to this flat part, if we go over here to our delta G place. So now it can easily run back across this flat part of the curve. That is, go through enolase phosphoglycerate mutase, phosphoglycerate kinase, GAPDH, taking advantage of the high NADH/NAD+ ratio that would exist when you're doing gluconeogenesis-- triosephosphate isomerase, aldolase. This generates a DHAP and a glyceraldehyde 3-phosphate that you can then combine to give a fructose 1,6bisphosphate. Now we get back here to now we have this steep part there. But rather than going through FBP, we can just have a phosphatase, fructose 1,6-bisphosphatase that releases the phosphate. Now, we have fructose 6phosphate. That fructose 6-phosphate can then go through glucose 6-phosphate isomerase and generate glucose 6phosphate. That now gets back to this other steep hill. So this is relatively flat. Now, you have this steep hill around hexokinase. But rather than coupling it to adding to ATP-ADP hydrolysis, you just remove the phosphate with glucose 6-phosphatase. And in the end, now we have a pathway using these four extra enzymes that give us a way to start at pyruvate and run the opposite direction and generate glucose. And you can do so in a way that is thermodynamically favorable. In other words, we can start with 2 pyruvate and use 4 ATP-- so and ATP to turn pyruvate twice into oxaloacetate. So that's two. And then another ATP at phosphoglycerate kinase to go backwards there to go from-- to run that reaction backwards, plus 2 GTP. Those 2 GTP are effectively the equivalent of an ATP. So this would be 6 ATP equivalents. Why are the GTPs equivalent of ATP? I just want to point out that cells have enzymes that basically can interconvert ATP and GDP or, really, any nucleoside triphosphate and diphosphate to GTP plus ADP. This has a delta G 0 prime that is effectively 0. And so cells basically match their ATP to ADP ratios with any other ratios of nucleoside triphosphates and diphosphates because of this reaction. And so that's why the two GTP are roughly an ATP equivalent. So we'll just call it 6 ATP plus 2 NADH to reverse the GAPDH step. That gives us 6 ADP plus 2 NAD+ plus 6 inorganic phosphate plus a glucose. The delta G 0 prime for this entire coupled pathway is on the order of negative 9 kcals per mol, with the delta G less than 0, equilibrium lies to the right. Net synthesis of glucose is favorable. And so if we just think about this in ATP terms, remember, when we discussed glycolysis, you harvest net two ATP from conversion of glucose into two pyruvate. If we reverse the reaction doing gluconeogenesis, in ATP terms, we have to invest six ATP to turn that pyruvate back into glucose. Makes sense. We didn't make a perpetual motion machine. Both pathways are favorable. But it costs more energy to make a glucose than we can get out by burning the glucose. I should say, the energy isn't just the ATP. There's also this NADH.

~16:23That NADH that's produced from glycolysis also is storing energy, if you will, from oxidation and an NADH/NAD+ ratio, and you're consuming that going in the opposite direction. You only do gluconeogenesis if you have a high ATP:ADP ratio, of course, and a NADH/NAH+ plus ratio-- that is, if you have it under the right conditions to actually run this pathway and have it make sense for the cell. All right. Now I want to discuss, briefly, how these enzymes in gluconeogenesis work. I'm not going to spend time on fructose 1,6-bisphosphatase or glucose 6-phosphatase. As I said before, this is just hydrolysis of that phosphate-alcohol bond. That's relatively straightforward. But I do want to spend time on pyruvate carboxylase and PEP carboxykinase because this introduces a new reaction, carboxylation. But it also introduces a new concept in metabolism that is actually quite important in eukaryotic cells. And that is this issue of so-called compartmentalized metabolism. And, of course, you've learned in introductory biology that a big difference between eukaryotes and prokaryotes is eukaryotes have all of these organelles-- that is, these membrane-bound structures within cells that carry out various functions. So one reason that it's useful to have these different membrane-bound organelles is it creates different compartments within the cell. What does that mean? Well, if you have different compartments, that means you can create different conditions within each compartment. And that's very important for metabolism. Well, why is that important for metabolism? Well, because, remember, whether or not reactions are favorable depend on delta G, which, of course, is dependent on the equilibrium constant, which is related to delta G 0 prime but also to the ratio of the products and reactants within that compartment. And so by having different compartments, you can have different ratios of metabolites-- say, a different ratio of ATP to ATP or a different ratio of NADH to NAD+. Having those different ratios means that because delta G is proportional to the ratio of the reactants over the products-- I'm sorry-- products over reactants, by having different ratios of things like ATP and ADP, NAD, NADH, you can make different reactions more or less favorable depending on which compartment in the cell that they're located. Now it turns out that glycolysis and most of gluconeogenesis takes place in the cytosol of eukaryotic cells. The cytosol is basically the space inside the cell that's not inside another organelle. However, pyruvate carboxylase is a reaction that takes place in mitochondria because mitochondria have a particularly high ATP:ADP ratio. And it turns out, this helps favor the pyruvate carboxylase reaction. And so we'll talk a lot about reactions in the site cytosol versus the mitochondria. But basically, here's glucose to PEP. Whether you do that in one direction by glycolysis or the other direction by gluconeogenesis, that happens in the cytosol as does turning that PEP into pyruvate. But if you're going to reverse the reaction, eukaryotes take advantage of a different compartment, the mitochondria, with a high ATP to ADP ratio. And basically, in the mitochondria, turn that pyruvate into oxaloacetic acid-- that is, carry out the pyruvate carboxylase reaction. And then that pyruvate carboxylase reaction generates oxaloacetic acid, and then PEPCK can, either in the mitochondria or in the cytosol, generate PEP. And so here's a place where one can run one reaction-- PEP to pyruvate of t make ATP in the cytosol-- but a different reaction-- pyruvate carboxylase to turn pyruvate into oxaloacetic acid that would then be used for PEPCK to turn that back into PEP and have that take place in the mitochondria.

~19:39I want to point out, just so for MCAT exams and things like that, PEPCK is classically defined as a cytosolic activity, at least in terms of gluconeogenesis. Although, there is a mitochondrial isoform of PEPCK. And it's at least somewhat debated. There's some evidence that, at least, in some tissues, that might be important for gluconeogenesis as well. And so that's why I draw it as PEPCK using oxaloacetate to PEP either in the mitochondria or in the cytosol because that's an area of active investigation right now. All right. Let's start by talking about how does pyruvate carboxylase work? This is an example of a carboxylase reaction, that is, adding a CO2 group to a molecule to make a carbon-carbon bond. That purple CO2 is added to the CH3 of pyruvate of to make oxaloacetate. And how this happens, in many cases, uses a cofactor. And that cofactor is called biotin. Biotin, like many cofactors, is a vitamin. And this provides a useful functional group. In this case, enzymes that use biotin use ATP to basically drive CO2 addition to biotin. And then that CO2 on the biotin is then activated to be transferred to another molecule in a carboxylation reaction. All right. So what does biotin look like? I will draw it for you. So this, here, would be biotin. I've drawn it in the enolate form of biotin. I'll draw it in a different way in a second. Biotin is typically bound to a lysine molecule in the active site of an enzyme that uses this. And so this, here, is basically a peptide bond between the terminal amino group, the epsilon amino group of the side chain of a lysine in a residue that basically makes a peptide bond to link biotin into the active site of the enzyme that's using it. Biotin is often drawn in the keto form. And the active part of biotin is basically this top part of the molecule. And so to draw it to see differences between the enolate and keto form, I just want to show that quickly. I drew it in the enolate form because it's easier to see, I think, how it-- so this, here, would be the keto form where biotin is drawn most commonly. And basically, the reaction, the way biotin picks up a CO2 is as follows. And that is CO2 can exist particularly under basic conditions as CO2 or as-- this is bicarbonate. OK. So bicarbonate-- turns out, some enzymes use CO2 directly. Some enzymes use bicarbonate. It turns out that pyruvate carboxylase uses bicarbonate. So biotin bound in the active site of pyruvate carboxylase will utilize bicarbonate in the following way. And I'll just point out that the pH of the mitochondria is also more basic, which also helps this pyruvate carboxylation reaction. All right. So what happens is this bicarbonate is phosphorylated by ATP. This phospho intermediate can then react with this active site here of biotin to release the phosphate. And now, you now have this activated CO2 group that's attached to the biotin. Here's pyruvate drawn in the enol form. You can look back in your notes about how to interconvert it between keto and the enol form. I've shown that several times. And then this, then, can end up adding this CO2 to the end of pyruvate to generate oxaloacetic acid plus regenerate the biotin cofactor in the right form. It'll be the keto form, which can then get back to the enol form. And it's ready to do another catalytic cycle. And so, basically, pyruvate carboxylase uses ATP to produce this carboxylate of biotin. And then uses that to add the CO2 to pyruvate to generate oxaloacetic acid. Now I want to point out that oxaloacetic acid is both an alpha keto acid like pyruvate was. And it's a beta keto acid. And so this ketone is alpha to that carboxylic acid. And it's beta to that carboxylic acid. So it's both an alpha and a beta keto acid. And it turns out that the way PEPCK works is it takes advantage of the favorable decarbonization of a beta keto acid, which we talked about last time, along with GTP to phosphorylate pyruvate and trap it in the enol form.

~23:33And so this is how PEPCK works. So I'm going to draw GTP in this very stylized way-- so guanine with three phosphate groups. There's oxaloacetate. That decarboxylates-- takes advantage of this being a beta keto acid to decarboxylate and add the phosphate. And that's how we can generate PEP. OK. Great. And so that's how you can build a different pathway that uses ATP and GTP for energy input in order to run glycolysis in the opposite direction-- gluconeogenesis. All right. So how is gluconeogenesis regulated? Well, just like the principles we talked about in glycolysis, it works in a way that makes sense. And so you can guess that the steps that are going to be regulated are exactly the ones that you might guess. They were the same steps that were regulated running glycolysis. It's going to be these big changes where energy changes occur across the pathway, which are also the entrance and exit of the pathway. And basically, regulation has to be reciprocal. So in glycolysis, you want to increase glycolysis under conditions where you need to produce ATP. And you want to decrease glycolysis if you have enough ATP or enough of another downstream product, such as citrate. All right. Well, gluconeogenesis is going to be exactly the opposite. You certainly don't want to do gluconeogenesis if you need energy release. If you need ATP, you don't want to run gluconeogenesis. However, you want to increase gluconeogenesis, if the cell has ATP excess, you also want to do it if you have excess of other products like citrate because why go through the trouble of sending things down glycolysis if you have nowhere to put it. You might as well, instead, make glucose or shunt that glucose off to produce glycogen. And so let me just add that regulation here. And so major regulators of gluconeogenesis are as follows. And so high levels of citrate is going to stimulate fructose 1,6-bisphosphatase. So high levels of citrate will inhibit things coming through glycolysis and activate gluconeogenesis by acting at fructose 1,6-bisphosphatase to match the energy considerations. If you have high levels of AMP, energy charge is low. You want to stimulate glycolysis at phosphofructokinase. Similarly, that's going to inhibit gluconeogenesis at FBPase. And it turns out, high levels of ADP-- I'm sorry-- yeah, high levels of ADP, low energy charge, also inhibits PEPCK because you also don't want to try to generate PEP under those conditions. Great. All right. Now in animals, we also want to regulate glucose catabolism and production under signaling control. And this gets back to this other topic we talked about, such as the liver's job being to maintain a constant level of blood sugars. Most of you are very familiar with blood sugar control through diabetes. You've probably heard that this is under hormonal control, that the job of insulin is to lower blood glucose. And you also have hormones like epinephrine or glucagon whose job is to raise blood glucose. Why do you want to raise blood glucose? Well, if you have some kind of fight-or-flight response-- you see that lion, and you need to run away, your body has this adrenaline rush. That's what epinephrine is. That basically wants to supply more energy to your muscles, so that you can run away effectively. So it turns out, both insulin and glucose act on lots of tissues in the body. I'm going to focus on what they do in the liver. And that's because in the liver, this is a major organ that regulates blood glucose levels. So if you have excess glucose around, what do you want to do? You want to stimulate glucose uptake into cells, its metabolism, and its storage. And so insulin with high blood sugar basically wants to stimulate, taking glucose into the liver and storing it as glycogen.

~27:03If you want to run away from the lion, well, now you have epinephrine around. You want to make sure that as you're consuming that glucose in your blood that you're continually making more. And so you want the epinephrine to stimulate release of glucose either from gluconeogenesis or from the breakdown of glycogen. And both of these hormones act-- insulin and epinephrine-- act at a level where they can regulate enzymes of glycolysis and gluconeogenesis as well as entry and exit of glucose monomers in and out of glycogen. And so this is an example of how fructose 2,6-bisphosphate works. And so remember you learned from Professor Yaffe that epinephrine acts via cyclic AMP signaling, which turns on a kinase-- protein kinase A. And that protein kinase A can regulate enzymes that produce or break down, basically, these enzymes that produce or break down fructose 2,6-bisphosphatase which, in turn, regulates PFK activities such that you can match your need to burn that glucose versus doing gluconeogenesis in the case of the liver, so you can do enough gluconeogenesis in order to have glucose around for the body to use in that fight-or-flight response being driven by epinephrine. Now a major effective insulin and epinephrine signaling, though, is actually on release or storage of glucose molecules in glycogen. And I alluded last time in talking about the regulation hexokinase that glucose 6phosphate is basically the entry point to get glucose units in and out of glycogen. And I want to discuss how you add and subtract those glucose units into storage polymers. Now I'm going to discuss it in the context of glycogen storage in humans. But remember, plants actually store things also as glucose polymers. The chemistry is very similar. But, obviously, the regulation is very different.

~28:41Now you'll hopefully recall from past lectures that these storage polymers of glucose-- glycogen in humans, starch in plants-- are basically these alpha 1,4 linkages of glucose molecules. Remember, there was a nonreducing end and a reducing end of the molecule. So starch was a straight-chain polymer. Glycogen had these alpha 1,6 branch points that made this branch polymer, where there's lots of non-reducing ends and a single reducing end. And each of these non-reducing ends is a polymer of glucose molecules with this alpha 1,4 linkage that, at the branch point, has this alpha 1,6 linkage to make this long-chain branched polymer. Now when we discussed this at the time, I mentioned that this is useful because you have all these non-reducing ends that can be used to add or subtract glucose monomers. And that's great. This is a nice compact form of storage with lots of places to put or remove glucose from. You either store it quickly, insulin driving glucose storage-- or remove it quickly, epinephrine driving release of glucose from the glycogen. And so if we're going to have a way to add glucose polymers or break them down, those are two separate pathways, just like glycolysis and gluconeogenesis-- reciprocal activities, but we need two pathways to do it because delta G has to be less than 0 for each pathway to work. Also, we have to be able to regulate these separately because we don't want to have a futile cycle. Now the way you add and subtract glucose polymers to glycogen acts through glucose 6-phosphate being first converted to glucose 1-phosphate. So this, here, is alpha glucose 6-phosphate. Remember, it's alpha because I drew that hydroxyl at the one position pointing down. This, obviously, can enter glycolysis and come in from glucose via hexokinase. All right.

~30:19This is first converted via a mutase reaction to move the phosphate from the 6 to the 1 position of glucose. So this, here, is glucose 1-phosphate. The way the mutase reaction works is exactly the same analogous mechanism that I explained for phosphoglycerate mutase in glycolysis-- moves the phosphate in this case from the 6 to the 1 position of glucose. And then once you have that glucose 1-phosphate, that can be added to a non-reducing end of a glycogen polymer using an enzyme called Glycogen Synthase, GS. So that'll give me a glycogen molecule with one additional monomer added to the non-reducing end of the polymer. And you release that glucose back off via a different pathway, a different enzyme called phosphorylase. All right. Now the activity of these enzymes-- glycogen synthase and phosphorylase-- that is, the pathway to add glucose 1-phosphate glucose monomer to the polymer versus remove it from the polymer is under hormonal signaling control in animals. And basically, that hormonal signaling works as follows. So phosphorylase can be phosphorylated or dephosphorylated via a signaling enzyme, an enzyme like phosphorylase kinase, which is downstream of PKA, which is downstream of epinephrine signaling. OK. And so when phosphorylase is phosphorylated by phosphorylase kinase-- that has, has a phosphate group added is a signaling cascade-- it is in the active state. And when it's dephosphorylated, it's in the inactive state. Well, glycogen synthase also is subject to regulation by protein phosphorylation on the enzyme by a kinase. Except this has the opposite relationship. So when it is in the phosphorylated state, it is inactive. But it is in the non-phosphorylated state, it is active. And so protein kinase A, which is downstream of epinephrine, can both activate phosphorylation of phosphorylase and glycogen synthase. And that makes sense. You want to release glycogen glucose monomers from glycogen. You turn on, by phosphorylation, the pathway to release them. And you turn off the pathway to store them. Another kinase that phosphorylates to glycogen synthase is a kinase called GSK or Glycogen Synthase Kinase. This kinase is inhibited by insulin signaling. And so this is a negative of a negative. So effectively, a negative of a negative will keep it in the inactive state. And so insulin, by inhibiting the ability of GSK to put glycogen synthase in the inactive state, will make glycogen synthase active. And when you have high insulin around, that actives glycogen synthesis, and you store glucose monomers as glycogen. Get them out of the blood. OK. Now let's discuss the chemistry that allows us to do this glycogen synthase and phosphorylates reactions. Now as we go through this, what you will see is that when we break down the polymer, energy is released. So that's going to be breaking down a polymer. That's the right direction of entropy. And so that's going to be released. And storing it is going to require energy. That is, you're going to have to have some energy input to build a polymer. But nature actually does this in a way that is actually quite efficient. First, let's talk about how you store glucose 1-phosphate units by adding them to glycogen. So this uses energy input from UTP, which is an ATP equivalent for exactly the reason I described earlier. That is, because ATP plus UDP, like any other interconversion of nucleoside triphosphates and diphosphates is very close to equilibrium.

~33:26So the UTP to UDP ratio should be similar to the ATP the ADP ratio. And so UTP is really an ATP equivalent. But nature, for whatever reason, decided to use UTP here. And it generates a nucleoside sugar called UDP glucose. And so let me show you what that is. So here is glucose 1-phosphate. I will draw a stylized version of UTP here. And so this molecule with the UDP added to the glucose is UDP glucose. This will also generate a pyrophosphate. And that pyrophosphate can be hydrolyzed to generate two inorganic phosphates, effectively, like building any other polymer, by doing this downstream-- keeping that product of the first reaction low helps pull that reaction forward for polymer synthesis. This UDP glucose can now react with the non-reducing end. So this, here, will be the non-reducing end of the polymer. That's the OH group at the 4 position, at the nonproducing end. And so that then generates that alpha 1,4 bond to add to the polymer. So here, we have glycogen n plus 1 plus releases UDP. And so the net to add a glucose 1-phosphate to the non-reducing end of the growing glycogen polymer is net conversion of a UTP to a UDP. So that is one ATP equivalent to add glucose 1-phosphate to the end of the polymer. Now, of course, you had it to also have an ATP from hexokinase to capture that glucose 6-phosphate to begin with. But effectively, this is two ADP molecules to add a glucose to glycogen. All right. Now if we're generating starch, that's all we have to do, just build a long polymer. But if we're making glycogen, remember, glycogen has all of these branch points on them as well. And to do the branch points, you basically have to make this alpha 1,6 bond, which then gives you two new non-reducing ends that you can add more subunits to. The way nature does this is as follows. And so if I just draw this here in a stylized way, it turns out, once you get to about seven units here in the growing polymer-- so this is the reducing end, and this is the non-reducing end of the polymer. And so the polymer is growing by adding things to that end. Once you get about seven, there's an enzyme that will basically cleave this, and move it and make a new alpha 1,6 bond. OK. Here we go. We have our reducing and our non-reducing end and basically transfers these 7 units over. And now, we have two non-reducing ends that we can continue to grow the polymer from. You can think of this like the airplane analogy. When you load an airplane, at least the way that's most efficient, the people who are sitting in the back of the plane, they get on first. But then, they're the last people off the plane because you add basically to these non-reducing ends, and then you subtract from those non-reducing ends. And so the first glucose added, the one at the reducing end, is the last one that is going to be removed. All right. Now phosphorylase is the enzyme that allows you to do the opposite-- that is, to break that alpha 1,4 bond and release monomers. And it's called phosphorylase because it uses phosphate to break that alpha 1,4 bond, taking advantage of the fact that breaking down the polymer is favorable and actually releasing the glucose from it again is a glucose 1-phosphate. And so at the non-reducing end of the molecule-- so this, here, would be the non-reducing end of the glycogen polymer. You basically have a phosphate molecule that takes that off, so you have a glycogen n minus 1 plus a glucose 1 phosphate, which can now undergo a mutase reaction back to glucose 6-phosphate that could then be released from the cell if it's a liver, or it could be burned in glycolysis.

~37:02Now, obviously, if you're doing breakdown, that is, your chewing back from non-reducing ends, eventually, you're going to hit a situation where you come to where there's just a nub here with an alpha 1,6 bond there. That is, chew it back till you hit this single monomer with a non-reducing end here to this alpha 1,6 break point. Turns out, you just cleave off that nub, if you will. And so that nub is then just released as free glucose. So other than release of that nub, what it means is that you actually basically get no ATP to actually break down in the phosphorylase reaction. So the net cost to store a glucose 1-phosphate molecule is one ATP via the UTP to UDP conversion in the glycogen synthase reaction. And you get that one glucose 1-phosphate back. And so two ATPs to put a glucose tin glycogen. And then you get it directly back out as a glucose 1-phosphate. That's a phosphorylated glucose that can then enter glycolysis. So that's one less phosphate you need to spend on glycolysis. And so it's incredibly efficient to store glucose in that way. And so it's actually slightly greater than one ATP to store it as glycogen because you lose in ATP with that nub removal at the branch polymer. But for all intents and purposes, it costs one ATP to store your glucose as glycogen, which is pretty amazing. All right. Now before I leave the topic of sugar storage, I want to make some comments on some sugars that are stored in forms other than glucose. And want to talk about these because these sugars are, of course, important parts of our diet. And so as we've mentioned in prior lectures, two of these sugars are sucrose and lactose. So remember, sucrose is a disaccharide of glucose plus fructose. And lactose is a disaccharide of glucose plus galactose. So those are storage forms of sugar-- sucrose in the case of plants, lactose in the case of milk made by mammals. And so to break those sugars down, basically, you break the disaccharide. Now, you have the glucose molecules you know how to handle. That's glycolysis. But I want to spend a little bit of time how you deal with the fructose and the galactose that you get out from these sugars because this actually relates to some things that I'm sure you've all read about in the news. For instance, fructose as a sugar in our diet is actually quite controversial. Lots of stuff out there that says fructose was cooked up in the devil's kitchen and is this evil thing. There's actually others who argue that, oh, fructose is the same as glucose, the same number of calories. There's both sides of the debate. Both sides are actually making factual claims. And I want you to understand the biochemistry, so you can actually judge for yourself who's right in those claims. All right. So let's start with talking about how you metabolize fructose. And so fructose is first captured by phosphorylation to generate fructose 1-phosphate. This is carried out by an enzyme called ketohexokinase. Ketohexokinase is an enzyme this present in your gut and your liver. And so fructose in the diet, from breaking down sucrose or from high-fructose corn syrup, whatever, is captured with ketohexokinase to make fructose 1-phosphate. Turns out that phosphofructokinase, our old friend from glycolysis, is less efficient than ketohexokinase but can also carry out this reaction. And remember, what is it do in glycolysis? It adds a phosphate to the 1 position of fructose 6phosphate. Well, it can also add a phosphate to the 1 position of fructose give you fructose 1-phosphate.

~40:24OK. Once you have this fructose 1-phosphate, which, remember, is not in glycolysis, it can be a substrate for the glycolytic enzyme aldolase. What does aldolase do in glycolysis? It splits FBP into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Well, here, you only have a phosphate on the 1 position. And so if you look back at how aldolase works, what you'll see is that if aldolase acts on fructose 1-phosphate, you'll generate a dihydroxyacetone phosphate. That phosphate will go to the dihydroxyacetone half, which, of course, is perfectly good substrate for glycolysis. The other product of aldolase acting on fructose 1-phosphate is just glyceraldehyde without the phosphate on it. And then there's another enzyme that will phosphorylate glyceraldehyde to generate glyceraldehyde 3phosphate, which can then also, of course, enter glycolysis. And so fructose is a hexose just like glucose. You generate two trioses-- DHAP and glyceraldehyde 3-phosphate. That costs you two ATP to do it, exactly the same cost is what you spent to get glucose to make DHAP and glyceraldehyde 3-phosphate. And so people are absolutely right when they say metabolizing fructose has exactly the same caloric cost as metabolizing glucose-- two ATPs to get it into the trioses. And then whatever you get out of it, you get out of it two ATPs, four ATPs-- so net two ATPs if you just do fermentation, more if you do complete oxidation. But the bottom line is that from that standpoint, you get exactly the same number of calories from burning glucose versus fructose. However, I want to point out that the regulation will not be the same. And that's because in this fructose metabolism, you will notice that there was no glucose 6-phosphate and no FBP generated. That means you are avoiding all of the feedbacks that control how much glucose you send to glycolysis-- first to glycogen. Remember, that's what glucose 6-phosphate does, acting on trapping sugar. And more importantly, you're actually not using phosphofructokinase in a way to fit with the rest of the feedback regulation. I also show that here on this slide. It's basically a better version of what I drew up here. And I now want to point out this storage of citrate and this feedback regulation of citrate. So we'll talk about the specifics of this more in other lectures. But it turns out, citrate is the precursor for how one stores carbon as fat. And so if you're putting fructose glucose into the system and citrate is too high, it'll shut off phosphofructokinase and stop carbon coming down the system, shunting it instead to glycogen or just saying don't give me any more glucose at all. If I'm doing the same thing with fructose, those feedbacks don't exist. Citrate doesn't feed back on any of the enzymes that are regulated for fructose metabolism. And so the idea is carbon keeps getting dumped into citrate, which ultimately ends up as fat. And this really underlies, at least on a theoretical basis, why there may be a connection between fructose and obesity or metabolic syndrome. I've posted articles on Stellar, one from The Popular Press, one from a scientific journal that discusses the controversy, discusses this. But now, at least, you have the background to draw some of your own conclusions. OK. That's fructose metabolism. What about galactose metabolism? Well, galactose metabolism interfaces with, effectively, glycogen metabolism or, at least, glucose 1-phosphate-or sorry, UDP glucose and does so in a way that is a little bit non-intuitive. But it's actually very important to understand galactose metabolism, at least for those of you who are looking to go to medical school.

~43:42It turns out that galactose metabolism and issues with metabolizing galactose are what underlie a set of rare genetic diseases leading to a set of conditions called galactosemias. I guess, if you're a pediatrician, you may encounter these. If you're not, these are quite rare. Although med schools love to ask about these, and certainly, they're very popular on things like board exams. So it's something you should know about. And so I just want to briefly introduce galactose metabolism to you.

~44:09So this is galactose. Like all sugars, galactose is initially trapped by phosphorylation. It's phosphorylated on the 1 position to trap it, just like fructose. And so this is galactose 1-phosphate. Remember, galactose is an epimer of glucose. So it differs from glucose here by the stereochemistry of the hydroxyl group at this 4 carbon. And so the way galactose, once it's trapped as galactose 1-phosphate is metabolized is it will react with UDP glucose. So here is UDP glucose from glycogen metabolism drawn in a very stylized way. That will generate a UDP galactose and a glucose 1-phosphate, which, of course, can enter glycolysis, can be stored as glycogen, et cetera. This UDP galactose can then be turned into UDP glucose via an epimerase reaction. And so remember, the epimerase reaction needs to change the stereochemistry of the hydroxyl group at this 4 position, such that it's pointing up in galactose or pointing down in glucose. The way epimerases work is that they require a cofactor. That cofactor is NAD. and they work in the following way. And so if I just draw, here's the 4 position of galactose, the 4 carbon of galactose, with the hydroxyl group pointing up, an epimerase can basically remove a hydride from the bottom face of the molecule, transfer those two electrons to NAD to generate NADH. Now, you get this lactone intermediate at the 4 carbon. And then you can have two electrons from an NADH can be added to the opposite face of the molecule that regenerates an NAD+. And now, we've effectively changed the stereochemistry at the 4 carbon to now be pointing such the hydroxyl points down, and that's glucose. And so the way epimerases work is they basically remove and add electrons from different faces of the pyranose ring. And that changes the stereochemistry of which direction the hydroxyl group is pointing. And so effectively, this is how we metabolize all kinds of different sugars, either putting them in glycogen or other starch, some other storage carbohydrate that we can get them later, and then use them in glycolysis as a way to drive glucose oxidation as a way to release energy and keep ATP high in the cell. All right. Now I want to shift topics and begin to discuss how we can oxidize pyruvate from glycolysis to make CO2 as an alternative to how we can release energy from carbohydrate oxidation rather than just use fermentation. Now you'll see that the enzymes and ways that we do this is going to also be how we make citrate. And so this isn't just about releasing energy. It's also about storing energy because you'll see, citrate is a precursor for storage as fat. The reactions downstream of pyruvate oxidation also make lots of other useful intermediates that can be used to synthesize other things cells need-- amino acids, nucleic acids. And so this pathways we're going to discuss are going to come up over and over again throughout the course. Now to facilitate this discussion, I'm really going to at least initially focus on catabolism.

~46:58And so just to remind you, if we do glycolysis by turning glucose pyruvate, this involves oxidation. So we need to dispose of those electrons somewhere. In other words, that NADH has to be regenerated NAD. Fermentation of pyruvate into something like lactate is a way to do that and allows net ATP production from glycolysis without the need for any oxygen. However, if we're going to completely oxidize that pyruvate CO2 we're going to generate many more electrons as waste that we have to put somewhere. Oxygen is a particularly good electron acceptor. And so we can reduce oxygen to water as a way to deal with that electron waste, and that allows us to, just burning wood, release lots of energy from glucose by transferring those electrons ultimately to oxygen. And of course, that same process-oxygen to water-- if we're using the pyruvate to fully oxidize it, also is necessary to regenerate the NAD to run glycolysis. Now ultimately, the series of reactions that allow chemically conversion of pyruvate to CO2 is called the TCA cycle for try Tricarboxylic Acid Cycle or sometimes referred to as the Krebs cycle and also is referred to as the citric acid cycle-- so TCA, Tricarboxylic Acid Cycle, Krebs cycle, citric acid cycle-- all synonyms for the same thing. What these cycles are is basically a way to take two carbon units. And those two carbon units can come from the oxidation of pyruvate or from the oxidation of lots of other fuels. And those two carbon units are combined with four carbon units. You'll see oxaloacetate is the four-carbon unit-- oxaloacetate gluconeogenesis. That oxaloacetate could come from the pyruvate-carboxylase reaction. Or it could come, as you'll see, from the TCA cycle itself. This generates a six-carbon molecule, which is citrate. Citrate is a tricarboxylic acid, hence the citric acid cycle or the tricarboxylic acid cycle. And then those six carbon units are reoxidized, releasing two CO2 molecules, back to four-carbon oxaloacetate. And hence, this is a cycle. Now this cycle was initially described by Hans Krebs, who predicted the cycle largely based on chemistry-- one of the most amazing feats in the discovery of metabolism. Turned out, he was right. And thus, it's also referred to as his name, the Krebs cycle. Now lots of oxidation is happening in this cycle. You're oxidizing the molecule, releasing CO2. That means lots of energy is released. Those electrons ultimately will be transferred oxygen. And so we can use this to make lots of ATP and do lots of other work. Where are these reactions all occur is in the mitochondria, specifically in the matrix of the mitochondria. Remember, the mitochondria, from cell biology, is a double-membrane organelle. The innermost part of the mitochondria is referred to as the matrix. And these have conditions that favor pyruvate carboxylase reaction, as I talked about, but also favor other oxidation reactions, such as those that occur in the TCA cycle. And so next time, what I will start with is by being more explicit about what chemistry is happening to allow these two carbon units to combine with oxaloacetate 4-carbon to generate citrate and go over the reactions that then allow you to run a cycle to regenerate oxaloacetate, that is, run the TCA cycle as a way to oxidize carbon. Thanks.

3.2糖原的合成与分解及双刃剑调控：为什么磷酸化同时开启分解又锁死合成？

一、糖原的树状分支结构与“飞机登机”模型

当餐后血糖升高时，机体不能让游离葡萄糖在细胞内无限制堆积——否则细胞内渗透压会急剧飙升，把细胞直接涨破！因此，动物将多余的葡萄糖聚合成高分子同多糖——糖原（Glycogen）（主要储存在肝脏和骨骼肌细胞质的糖原颗粒中：肝糖原占肝湿重 7%～ 10%，用于维持血糖；肌糖原占骨骼肌湿重 1%～ 2%，但因全身肌肉总量大，肌糖原总储量约为肝糖原的 2～3 倍）。

糖原与植物支链淀粉结构相似，均由 α-D-葡萄糖通过 α-1,4-糖苷键连接成主链，并通过 α-1,6-糖苷键形成分支 2025·填空，但糖原的分支密度比支链淀粉高得多（糖原每 8～12 个葡萄糖残基就有一个 α-1,6 分支，而支链淀粉每 24～30 个残基才有一个分支）。

二、糖原的分解（Glycogenolysis）：磷酸解的化学智慧与三酶接力

为什么糖原分解不直接用水（H_2O）去水解 α-1,4-糖苷键（像消化道里的淀粉酶那样），而是专门用无机磷酸（P_i）去进行磷酸解（Phosphorolysis）？Vander Heiden 教授算了一笔精明至极的能量账：

1. 第一步：糖原磷酸化酶（Glycogen Phosphorylase, GP）催化非还原端磷酸解（分解限速酶）
   糖原_{(n 个残基)} + P_i ──(糖原磷酸化酶（辅基 PLP）)──► 糖原_{(n-1 个残基)} + α-D-1-磷酸葡萄糖（G-1-P）

• 辅酶与催化机理：糖原磷酸化酶是糖原分解的限速酶（关键酶），以磷酸吡哆醛（PLP，维生素 B₆ 活性形式）为必需辅基，直接生成的产物是 1-磷酸葡萄糖（G-1-P，保留 α 构型） 2026·单选。
• 注意：PLP 在转氨酶和脱羧酶中靠醛基形成 Schiff 碱充当“电子槽”，但在糖原磷酸化酶中，PLP 的醛基早已与酶的 Lys680 共价锁定，真正参与催化的是 PLP 的 5'-磷酸基团！ 它作为广义酸碱催化剂，向底物无机磷酸 P_i 提供质子并协助其进攻 α-1,4-糖苷键，经由一个半椅式氧鎓离子（oxonium ion）过渡态完成磷酸解，且因酶活性口袋完全排斥水分子而绝不发生水解！
• 为什么磷酸解比水解优越得多？ ① 省下 1 分子 ATP：水解产生的是游离葡萄糖，要进入糖酵解还得耗费 1 分子 ATP 被己糖激酶磷酸化；而磷酸解直接用廉价的无机磷酸 P_i 换来了带磷酸基团的 G-1-P，变位成 G-6-P 毫不费力（不耗 ATP），因此糖原中的 1 个葡萄糖残基走糖酵解净赚 4 - 1 = 3 ATP（比游离葡萄糖多赚 1 ATP）！② 负电荷锁闭效应：G-1-P 在生理 pH 下带负电荷，无法穿透细胞膜外漏，被牢牢锁在肌肉细胞内供能！
• 立体阻碍与极限糊精：糖原磷酸化酶是个“大块头”，当它沿着非还原端一路向内啃食，距离 α-1,6 分支点还剩 4 个葡萄糖残基时，由于分支空间位阻卡住，磷酸化酶便寸步难行，留下的这种残余分支核心称为极限糊精（limit dextrin）。

1. 第二步：糖原脱支酶（Debranching Enzyme）的双功能拆解
   为了清除分支路障，细胞派出了一条肽链上兼具两种催化活性的双功能糖原脱支酶：

• ① 寡聚-(α1,4→α1,4)-葡聚糖转移酶活性（先搬家）：将极限分支上最外侧的 3 个葡萄糖残基作为一个整体切下，转移并连接到相邻主链的非还原端 C4-OH 上（形成新的 α-1,4 键），只在分支点留下孤零零的 1 个以 α-1,6 键相连的葡萄糖残基（Vander Heiden 教授称之为“nub 小肉刺”）。
• ② 淀粉-α-1,6-糖苷酶活性（后切除）：用一分子 H_2O 将这个单独的 α-1,6 键水解（注意：这里是水解而非磷酸解！），释放出 1 分子游离葡萄糖！
• 由于糖原平均每 8～12 个残基有一个分支点，因此糖原彻底降解的产物中，约 90% 是 1-磷酸葡萄糖（G-1-P），约 10% 是来自脱支酶水解产生的游离葡萄糖！清除了分支后，糖原磷酸化酶又能继续畅通无阻地向内磷酸解。

1. 第三步：磷酸葡糖变位酶（Phosphoglucomutase, PGM）催化磷酸移位
   1-磷酸葡萄糖（G-1-P） ↔(磷酸葡糖变位酶（Ser-P、1,6-二磷酸葡糖引物）) 6-磷酸葡萄糖（G-6-P）
   Vander Heiden 教授特别提醒大家回忆糖酵解第 8 步的磷酸甘油酸变位酶：两者的“有借有还”机制如出一辙！不同的是，磷酸甘油酸变位酶活性中心是磷酸化组氨酸（His-P），而磷酸葡糖变位酶活性中心是磷酸化丝氨酸（Ser-P）：酶先把自身 Ser 上的磷酸基借给底物 G-1-P 的 C6-OH，形成 1,6-二磷酸葡萄糖（G-1,6-BP）中间体，随后再收回 C1 位的磷酸基，释放出 G-6-P 并使酶恢复磷酸化状态。生成的 G-6-P 在骨骼肌中直接进入糖酵解供能，在肝脏中则进入内质网被 G-6-Pase 水解为游离葡萄糖释放入血。

三、糖原的合成（Glycogenesis）：为什么必须借道 UDPG 与生糖原蛋白引物？

尽管体外高浓度 G-1-P 下糖原磷酸化酶反应可逆，但在活细胞内 [P_i]/[G-1-P] 比值高达 30～ 100:1，热力学决定了糖原磷酸化酶在体内只能单向催化糖原分解！要想逆着熵增方向把葡萄糖单体聚合成糖原，细胞必须另辟一条由高能核苷酸 UTP 驱动的合成通道：

1. 第一步：UDP-葡糖焦磷酸化酶（UDPG Pyrophosphorylase）活化糖基供体
   1-磷酸葡萄糖（G-1-P） + UTP ──(UDP-葡糖焦磷酸化酶)──► UDP-葡萄糖（UDPG） + PP_i (Δ G'^° ≈ 0 kJ/mol)
   PP_i + H_2O ──(无机焦磷酸酶)──► 2 P_i (Δ G'^° = -19.2 kJ/mol)

• 反应机理：G-1-P 的磷酸氧原子亲核进攻 UTP 的 α-磷原子，脱去焦磷酸（PP_i，由 UTP 的 β,γ-磷组成），生成糖核苷酸——尿苷二磷酸葡萄糖（UDPG），它是动物体内合成糖原、寡糖和多糖时葡萄糖的直接活性供体 2010/2013/2016/2018·单选2011/2025·填空（而植物合成淀粉时的糖基供体则是 ADPG！）。
• 为什么生物界在合成多糖、核酸、脂酰-CoA 和氨酰-tRNA 时，总是偏爱释放焦磷酸（PP_i）？ 因为第一步缩合反应本身的 Δ G'^° ≈ 0，但细胞质中无处不在的无机焦磷酸酶（Inorganic pyrophosphatase）会立即将产物 PP_i 水解为 2 P_i 并释放出 -19.2 kJ/mol 的自由能，依据勒夏特列原理将产物浓度抽空，像一台强力抽水机一样把整个聚合活化反应变为不可逆的单向通道！

1. 第二步：生糖原蛋白（Glycogenin）自糖基化引物与糖原合酶（Glycogen Synthase, GS）延伸主链
   UDPG + 糖原_{(n 个残基)} ──(糖原合酶（GS，合成限速酶）)──► 糖原_{(n+1 个残基)} + UDP (Δ G'^° = -13.4 kJ/mol)

• 引物铁律：糖原合酶是糖原合成的限速酶，但它就像 DNA 聚合酶一样，绝对不能从零开始把两个游离葡萄糖拼在一起，必须依赖至少含 4 个以上 α-1,4 葡萄糖残基的寡糖链作为引物（Primer）！
• 那么第一个引物从哪来？来自位于每个糖原颗粒最中心的生糖原蛋白（Glycogenin，又称糖原核心蛋白）！它既是引物支架，自身又具有葡糖基转移酶活性：能以 UDPG 为供体，将第 1 个葡萄糖残基以糖苷键共价连接到自身 Tyr194（酪氨酸）的酚羟基上，随后自我催化连续接上约 8 个 α-1,4 连接的葡萄糖残基，构成短短的初始引物链；此后糖原合酶（GS）才正式接手，把 UDPG 上的葡萄糖基不断加到引物链的非还原端 C4-OH 上！释放的 UDP 则由核苷二磷酸激酶消耗 1 分子 ATP 重新磷酸化为 UTP（UDP + ATP → UTP + ADP）。

1. 第三步：糖原分支酶（Branching Enzyme）构建 α-1,6 分支
   糖原合酶只会傻傻地延长 α-1,4 直链。当一条直链延长到至少 11 个葡萄糖残基以上时，糖原分支酶（全称：淀粉-[1,4→ 1,6]-转糖基酶）便会切下非还原端约 6～7 个残基的一段寡糖链，将其转移到同一条链或相邻链内部某个葡萄糖残基的 C6-OH 上，形成一个全新的 α-1,6-糖苷键分支（且新分支点与旧分支点之间至少相距 4 个残基），从而让非还原端的数量呈指数级倍增！

四、双刃剑共价修饰与级联放大：肾上腺素/胰高血糖素 vs 胰岛素

既然糖原分解（GP）和糖原合成（GS）的酶都在同一个细胞质颗粒上，细胞如何避免“一边耗 UTP 合成糖原、一边磷酸解糖原”的无效循环？答案是生物学中最经典的蛋白激酶/磷酸酶互反共价修饰与激素级联放大系统！

3D3D 调节：可逆磷酸化与糖原磷酸化酶 5 步级联放大蛋白激酶/磷酸酶可逆开关、cAMP 别构解离激活 PKA(R2C2) 及肾上腺素激活糖原磷酸化酶 10^5 倍级联放大↗

3D3D 分子：糖原磷酸化酶 b → a 与百万倍级联放大（五考大题）步骤 3：Ser14 磷酸化激活促糖原分解升血糖，同时 PKA 使糖原合酶磷酸化失活↗

除了激素共价修饰，肝脏与骨骼肌的糖原代谢还受到精妙的组织特异性别构调节（华农选择题与简答题极易深挖此处）：

1. 骨骼肌的“能量与钙信号传感器”：骨骼肌糖原只为自身收缩服务。当肌肉剧烈收缩时，一方面 ATP 水解产生大量 AMP，AMP 别构激活肌肉糖原磷酸化酶 b（而高浓度 ATP 和 G-6-P 则别构抑制磷酸化酶 b）；另一方面肌浆网释放大量 Ca²⁺，直接结合到磷酸化酶 b 激酶的 δ 亚基（即钙调蛋白 Calmodulin）上，无需磷酸化即可部分激活该激酶，将肌肉收缩的电-机械信号与糖原分解供能瞬间偶联！
2. 肝脏的“血糖浓度传感器”：肝脏的任务是为全身调节血糖，因此肝糖原磷酸化酶 b 完全不受 AMP 的别构激活！相反，游离葡萄糖是肝糖原磷酸化酶 a 的别构抑制剂：当餐后门静脉高浓度葡萄糖涌入肝细胞时，葡萄糖直接结合肝磷酸化酶 a 的别构位点，促使其构象由 R 态翻转为 T 态，把原本埋藏在内部的 Ser14-磷酸基暴露出来，让 PP1 迅速将其去磷酸化灭活为 b 型，同时解除对 PP1 的抑制，使糖原合成即刻启动！

五、MIT 7.05 L22 课堂专题：果糖与半乳糖如何进入代谢？为什么果糖更易诱发肥胖与脂肪肝？

在日常饮食中，我们摄入的蔗糖（葡萄糖 + 果糖）和高果糖玉米糖浆（HFCS）含有大量果糖（Fructose），乳制品中的乳糖（葡萄糖 + 半乳糖）则含有大量半乳糖（Galactose）。Vander Heiden 教授在 L22 用了整整两黑板来剖析这两种单糖的代谢入口，并揭示了一场著名的营养学论战：

1. 果糖代谢的“双轨制”与绕过 PFK-1 的“脂肪直通车”

媒体上有人把果糖称为“魔鬼厨房里熬出来的毒药”，也有食品工业界辩称“果糖和葡萄糖都是六碳糖，卡路里和耗能一模一样”。Vander Heiden 教授指出：双方说的都是事实，但关键差异不在能量账单，而在别构调控的彻底失灵！

• 在肌肉和脂肪组织中，果糖可被己糖激酶直接磷酸化为 F-6-P 进入糖酵解，受 PFK-1 正常调控。
• 但在肝脏（膳食果糖的主要处理器官）中，果糖走的是另一条专门通道 2025·简答：

1. 果糖激酶（Fructokinase，又称酮己糖激酶 Ketohexokinase）：消耗 1 分子 ATP，专一地在果糖 C1 位加上磷酸，生成 1-磷酸果糖（Fructose-1-phosphate, F-1-P）；
2. 1-磷酸果糖醛缩酶（即肝特异性的醛缩酶 B，Aldolase B）：将 F-1-P 直接裂解为 磷酸二羟丙酮（DHAP） + 游离甘油醛（Glyceraldehyde，不带磷酸！）（注：若先天缺乏醛缩酶 B，摄入果糖会导致 F-1-P 在肝内大量堆积、耗竭细胞内 P_i 和 ATP，引发致命的遗传性果糖不耐症）；
3. 丙糖激酶（Triose kinase，甘油醛激酶）：再消耗 1 分子 ATP，将游离甘油醛磷酸化为 3-磷酸甘油醛（GAP），与 DHAP 一起汇入糖酵解下段！

• 为什么过量摄入果糖比葡萄糖更容易导致肥胖和脂肪肝？ 算能量账单，1 分子果糖变成 DHAP + GAP 消耗 2 分子 ATP，与葡萄糖完全相同；但是在肝脏中，果糖经 F-1-P 直接裂解为磷酸丙糖，彻底绕过了糖酵解的总阀门——磷酸果糖激酶-1（PFK-1）！ 当你吃下大量葡萄糖时，一旦线粒体产生的柠檬酸和 ATP 偏高，就会强烈反馈抑制 PFK-1，并促使 G-6-P 转向合成肝糖原或停止摄取；而当你喝下含高果糖玉米糖浆的甜饮料时，果糖代谢根本不经过 G-6-P 和 PFK-1，柠檬酸和 ATP 的反馈刹车完全失效，源源不断的碳骨架像决堤洪水一样直冲丙酮酸、乙酰-CoA 和柠檬酸，最终在肝脏中无节制地合成脂肪酸与甘油三酯！

2. 半乳糖代谢（Leloir 途径）与差向异构酶的“双面氢负离子翻转术”

乳糖消化释放的 D-半乳糖（Galactose）是 D-葡萄糖的 C4 差向异构体（在 Haworth 透视式中，葡萄糖的 C4-OH 朝下，而半乳糖的 C4-OH 朝上）。细胞如何把这个朝上的羟基翻下来？2025·判断/简答

1. 半乳糖激酶（Galactokinase）：消耗 1 分子 ATP 将半乳糖 C1 磷酸化，生成 1-磷酸半乳糖（Gal-1-P）；
2. 1-磷酸半乳糖尿苷酰转移酶（GALT）：让 Gal-1-P 与糖原代谢中的老熟人 UDPG 发生尿苷酰基交换，生成 UDP-半乳糖（UDP-Gal）和 1-磷酸葡萄糖（G-1-P）（G-1-P 直接经变位酶变成 G-6-P 进入糖酵解或合成糖原！）；
3. UDP-葡萄糖-4-差向异构酶（UDP-glucose 4-epimerase）：将 UDP-Gal 重新翻转回 UDPG，完成催化循环！

• Vander Heiden 教授的黑板化学推演：差向异构酶如何在不打断碳骨架的情况下反转 C4 的手性构型？该酶活性中心紧密结合着一分子辅酶 NAD⁺！催化时，NAD⁺ 先从吡喃糖环的一侧（下方）夺取 C4 上的氢负离子（H^-），将 C4 仲醇氧化为平面的 4-酮基中间物（4-keto intermediate），自身暂时变成 NADH；紧接着，该 NADH 旋转到糖环的另一侧（上方），把氢负离子重新加回到 C4 羰基碳上，再生 NAD⁺ 的同时，完美地把 C4-OH 的空间朝向从朝上（半乳糖）翻转成了朝下（葡萄糖）！
• 临床联系（半乳糖血症 Galactosemia）：若婴儿先天缺乏 1-磷酸半乳糖尿苷酰转移酶（GALT），饮用母乳或牛奶后 Gal-1-P 和半乳糖醇（由醛糖还原酶还原产生）会在肝、脑和晶状体中大量积累，引发肝肿大、智力发育障碍和先天性白内障，临床上必须立即停喂乳制品改用无乳糖配方奶。

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🎧 MIT 7.05 课堂原声英文实录对照（本节关联 1 讲 · 完整逐段实录）点击展开 / 收起 ▾

🎬 MIT 7.05 Lec 22 · Gluconeogenesis, Glycogen & TCA Cycle I⏱ ~50:00

~00:00MITOCW | 22. Glucogenesis/Carbohydrate Storage/TCA Cycle I

~00:02PROFESSOR:

~00:02So last time, we discussed glycolysis as a pathway. And I just want to remind everybody that, like all pathways, glycolysis has to be favorable. That is, delta G across the entire pathway has to be less than 0. We spent a lot of time discussing the regulation of glycolysis. And I just want to revisit this quickly. And remember, we discussed that the steps that are regulated are those with the biggest, quote, unquote, bio-energetic cost. That is, the biggest drop in delta G is as molecules would proceed across the pathway. And those are shown here-- the hexokinase step, the phosphofructokinase step, and the pyruvate kinase step of the pathway. Now this is largely true of most pathways that we will consider, in that these regulated steps are often those with the biggest bio-energetic cost. And the reason for that is that it makes a lot of sense. Once you go through those steps, it's very difficult to go back.

~00:55Also, you'll note that these are the steps that regulate entrance and exit from the pathway. And this also is something we'll commonly see and other pathways and also makes some sense. Just to remind everybody, I drew up here a skeletonized version of glycolysis and the most important regulatory features of it. Just to remind people that initially, hexokinase is regulated by glucose-6 phosphate. Glucose-6 phosphate negatively regulates hexokinase. And that's because if the glucose-6 phosphate levels in the cell go up such that it can't be shunted to glycogen-- something we'll talk about today-- or be sent through glycolysis, it makes sense not to trap more glucose. Once glucose-6 phosphate enters the rest of the glycolytic pathway, the key step of regulation is phosphofructokinase. It, of course, will have positive feedback, saying, let's put carbon into the pathway. If energy charge low, AMP is high, activates it. This molecule fructose 2,6-bisphosphate that we talked about earlier today-- we'll mention again today-- can be involved in some other signaling inputs to allosterically regulate the enzyme. FBP can feed forward to activate pyruvate kinase. You want to match input with output. And, of course, if PEP builds up, you want to slow it down. If a downstream product-- citrate-- builds up, you want to slow it down. And of course, a major output of glycolysis is ATP. And if ATP is high, no reason to continue oxidizing glucose carbons. Now this regulation we discussed is often done at the allosteric level by controlling enzyme rates. That can be via a small molecule binding, as I've shown up here. And it could also be via signaling. And that's really a place where fructose 2,6-bisphosphate as a combination of signaling, and allosteric small molecules comes into play. That is, you can have signaling that controls the enzymes that generate fructose 2,6-bisphosphate, thereby creating an additional molecule that can act as allosteric regulator of the enzyme PFK to generate FBP and commit carbon to glycolysis.

~02:44Now we closed the discussion last time beginning to discuss how it is that organisms can make glucose. Obviously, this glucose carbon that is being oxidized to get ATP has to come from somewhere. We mentioned that plants make glucose during the day when they have plenty of excess energy from light from the sun but then use that glucose to do constant metabolism to maintain their ATP levels at night. And we as animals obviously take advantage of this by eating the glucose that ultimately was created by plants using the energy of the sun. We began to discuss an example of where us, as organisms, also make glucose. Of course, this is not net production of glucose. But for instance, if our muscle cells are working hard, lactate can build up. That lactate is excreted into the blood. That lactate can then be used by the liver to regenerate glucose. And one of the major jobs of the liver is to maintain a constant level of blood glucose to continue to supply fuel for various organs, including the muscle to be able to carry out the work that it needs to do. And so this whole process is called the Cori cycle. And a major job of the liver is thus generating glucose to maintain blood glucose levels. And the pathways used to do this is a pathway that we began to talk about last time called gluconeogenesis. And it's really a discussion of gluconeogenesis. That is, how can one build a pathway in plants, in the liver, wherever, as a way to generate glucose from something like lactate? Now, of course, we know from last time, lactate dehydrogenase can, obviously, it's a redox reaction to interconvert with pyruvate. And what gluconeogenesis really is it's a pathway that cells use to convert pyruvate or really anything else that can come into glycolysis-- and example being lactate. This, of course, can happen by the lactate dehydrogenase reaction. And then once it has that pyruvate, turn that pyruvate into glucose. That's really what we're talking about when we're discussing gluconeogenesis. Now how can we turn pyruvate into glucose? Well, we just discussed the whole pathway where we could turn glucose into pyruvate-- glycolysis. And so this is really the reverse of glycolysis. I said many times that all enzymes are reversible. So why can't we just run the pathway in the opposite direction? Well, hopefully, if you've been paying attention for the last several lectures, you realize there's two problems with doing that. The first is that, remember, why we're doing metabolism in the first place is to maintain a high ATP:ADP ratio under conditions so that the cell can use that ATP:ADP ratio to carry out otherwise unfavorable processes. And so thus, at a very high level, cells can't stop catabolism. Glycolysis or whatever has to continue to maintain ATP in the right range. And so now, if we're going to do something anabolic-- that is, build something, turn it into glucose, this has to happen in the background where catabolism has still happened. And so at least you need some kind of way to control both and prevent this from being some kind of futile cycle. Second, even if that ATP level is super high in the cell, it has extra energy, there is still a thermodynamic problem with just reversing glycolysis. And that is, remember, delta G has to be less than 0 for any pathway to work. And so it then stands to reason that if glucose oxidation to pyruvate, if this reaction is favored, as it is in glycolysis, that delta G is less than 0.

~06:03Well, under the conditions where that's occurring, if we want to then say, well, what about the reverse? Let's switch the signs-- pyruvate to glucose. We know that delta G is the same as it was in the opposite direction but, now, changing the signs from positive to negative or negative positive. And so, thus, delta G, for the reverse reaction, must be greater than 0 and, therefore, must be spontaneous-- is not spontaneous. And therefore says that we need to have a different pathway simply for energetic reasons if we're going to go in the opposite direction. Now oxidizing glucose releases energy. That's how we keep the ATP:ADP ratio high. It stands to reason then that we are going to have to put energy in if we're going to run in the opposite direction. And so if we're going to build a pathway from pyruvate to glucose, you can guess, we're going to have to add couple that pathway to ATP-ADP hydrolysis-- that is, couple it to something favorable to now allow this otherwise unfavorable reaction to happen. Now this energy, of course, in plants can come from the sun. We'll discuss that in great detail when we get to photosynthesis. However, for the sake of you and I, I guess, what it says is, if you think about this, if you release energy from glycolysis, it makes sense then you have to add energy if you're going to do gluconeogenesis. But remember, to satisfy all the laws of thermodynamics, the net energy loss to the universe is going to be-- must exist, which means that it is going to cost more energy to make glucose than you're going to get back from breaking it down. That just, hopefully, fits with your intuition. And it actually, I guess, is good news, because when are we going to do anabolism? When are we going to store all these excess calories as glucose? Well, we're going to do it if we overeat. And if we overeat, and it costs us energy in the form of calories to actually store that energy for later, I guess, it means that when we overeat, we don't have things quite as bad as it could have been. All right. Now let's get back to the how one can actually do this. So if we want to think, what is a pathway that we can build to get from pyruvate back to glucose? Well, we know we're going to have to burn some ATP to do it. But what are the issues with simply reversing glycolysis? Well, we can come back here and say, well, what was driving glycolysis in the first place? And it was these big steps of free energy change acts of hexokinase, phosphofructokinase, and pyruvate kinase. Where as many of these other reactions, at least under these standard conditions that are estimated to exist in cells, are relatively close to equilibrium. So there's really no problem with reversing glycolysis across here or across there. The real issue comes from getting around the hexokinase to phosphofructokinase and the pyruvate kinase step. These are the places where the most energy is released, if you will, when you're going through glycolysis. So those are the steps we're going to have to add energy in order to reverse build a pathway that goes in the opposite direction and allows you to start with pyruvate and end up with glucose. And so what we will find is that gluconeogenesis and glycolysis use many of the same enzymes. However, not all of the enzymes are the same because we need different enzymes if we're going to get around this hexokinase, phosphofructokinase, and pyruvate kinase steps of the pathway. And so for gluconeogenesis, we essentially need four new enzymes in order to get past those places. So we're going to need a new enzyme called glucose 6phosphatase. And glucose 6-phosphatase is going to allow us to get from glucose 6-phosphate and turn it back into glucose.

~09:41All right. We're going to need an enzyme fructose 1,6-bisphosphatase, which is going to allow us to take FBP and turn it back into fructose 6-phosphate. And then we're going to need two enzymes to get around the pyruvate kinase step. These are two enzymes called PEPCK and PC. That stands for phosphoenolpyruvate carboxykinase and pyruvate carboxylase. I'll discuss these in great detail in a minute. And these are going to allow us to get from pyruvate back to PEP. Also, essentially, I'll add them up here to this diagram. So to get back from pyruvate to PEP, two enzymes-pyruvate carboxylase and phosphoenolpyruvate carboxykinase to get from fructose 1,6-bisphosphate, fructose 6phosphate, an enzyme called FBPase. And to get from glucose 6-phosphate back to glucose, an enzyme called glucose 6-phosphatase. OK. Now, of course, if you think back, we're also going to need to balance electrons in the pathway. Remember, glucose to pyruvate is an oxidation reaction. That is, we're generating NADH. That means to go in the reverse direction, we need a source of NADH to reverse that GAPDH reaction. I just want to point out that you're only doing gluconeogenesis if cells have plenty of energy. What does plenty of energy mean? Well, that means the ATP:ADP ratio is high because you're doing lots of metabolism. But if you're doing lots of metabolism, and the ATP:ADP ratio's high, that also means the NADH/NAD+ ratio is high. And so you can think of the NAD regeneration by gluconeogenesis as being, for all intents and purposes, an alternative to fermentation, if you will, as a way to keep catabolism going. That is, you're only doing this if you have plenty of NADH around. And so dealing with that electron balance is less of an issue. OK. Now let's discuss briefly how some of these enzymes work. So glucose 6-phosphate and fructose 1,6bisphosphatase are very straightforward. Effectively, all they are is simply removing a phosphate. That's very favorable. Remember, when we did those reactions of hexokinase phosphofructokinase reactions and glycolysis, that, we had to couple otherwise unfavorable things-- adding a phosphate group to ATP hydrolysis. This is the reverse of that. And so the reverse of that, delta G is going to be less than 0. Delta G less than 0 means we just have a phosphatase there, just like you might have on a kinase enzyme. That's very straightforward. But there's a much bigger issue to get around this other step, this pyruvate kinase step. Remember, equilibrium strongly favored PEP to pyruvate conversion. That was one of the reasons why we could use that step to maintain a high ATP:ADP ratio in cells. Well, now, the issue becomes, how can we trap that phosphate back onto pyruvate while generating phosphoenolpyruvate? How one does that is an issue. Even if the ATP:ADP ratio is high, it's never high enough to reverse pyruvate kinase on its own. And so we need a new pathway with two steps in it. That's why there's two enzymes-- PC and PEPCK-- that are there to create a pathway where pyruvate to PEP now becomes favorable. That is, where delta G 0 prime is less than 0. That is, the equilibrium favors to the right. And under cellular conditions, this pathway can actually happen such that cells can net make glucose. Now these are two steps, both of which require ATP. And so you effectively get one ATP from PEP to pyruvate conversion. It costs you to ADPs or two ADP equivalents to go back from pyruvate to PEP, which makes sense. You should have to spend more energy to go upstream than you get back coming downstream. All right. Here's how this pathway works.

~13:03So this is pyruvate, the first reaction catalyzed by PC, which stands for pyruvate carboxylase. Couples ATP hydrolysis to addition of a CO2 that generates this intermediate, which is called oxaloacetate, often abbreviated OAA for Oxaloacetic Acid. That oxaloacetate is then decarboxylated. That is, the CO2 that is lost is removed-- that was added is removed. This reaction is coupled to GTP and is catalyzed by this enzyme, PEPCK PEP carboxykinase and uses that phosphate from GTP to trap pyruvate in the enol form as PEP or phosphoenolpyruvate. great. So once you've regenerated PEP, now that PEP gets to this flat part, if we go over here to our delta G place. So now it can easily run back across this flat part of the curve. That is, go through enolase phosphoglycerate mutase, phosphoglycerate kinase, GAPDH, taking advantage of the high NADH/NAD+ ratio that would exist when you're doing gluconeogenesis-- triosephosphate isomerase, aldolase. This generates a DHAP and a glyceraldehyde 3-phosphate that you can then combine to give a fructose 1,6bisphosphate. Now we get back here to now we have this steep part there. But rather than going through FBP, we can just have a phosphatase, fructose 1,6-bisphosphatase that releases the phosphate. Now, we have fructose 6phosphate. That fructose 6-phosphate can then go through glucose 6-phosphate isomerase and generate glucose 6phosphate. That now gets back to this other steep hill. So this is relatively flat. Now, you have this steep hill around hexokinase. But rather than coupling it to adding to ATP-ADP hydrolysis, you just remove the phosphate with glucose 6-phosphatase. And in the end, now we have a pathway using these four extra enzymes that give us a way to start at pyruvate and run the opposite direction and generate glucose. And you can do so in a way that is thermodynamically favorable. In other words, we can start with 2 pyruvate and use 4 ATP-- so and ATP to turn pyruvate twice into oxaloacetate. So that's two. And then another ATP at phosphoglycerate kinase to go backwards there to go from-- to run that reaction backwards, plus 2 GTP. Those 2 GTP are effectively the equivalent of an ATP. So this would be 6 ATP equivalents. Why are the GTPs equivalent of ATP? I just want to point out that cells have enzymes that basically can interconvert ATP and GDP or, really, any nucleoside triphosphate and diphosphate to GTP plus ADP. This has a delta G 0 prime that is effectively 0. And so cells basically match their ATP to ADP ratios with any other ratios of nucleoside triphosphates and diphosphates because of this reaction. And so that's why the two GTP are roughly an ATP equivalent. So we'll just call it 6 ATP plus 2 NADH to reverse the GAPDH step. That gives us 6 ADP plus 2 NAD+ plus 6 inorganic phosphate plus a glucose. The delta G 0 prime for this entire coupled pathway is on the order of negative 9 kcals per mol, with the delta G less than 0, equilibrium lies to the right. Net synthesis of glucose is favorable. And so if we just think about this in ATP terms, remember, when we discussed glycolysis, you harvest net two ATP from conversion of glucose into two pyruvate. If we reverse the reaction doing gluconeogenesis, in ATP terms, we have to invest six ATP to turn that pyruvate back into glucose. Makes sense. We didn't make a perpetual motion machine. Both pathways are favorable. But it costs more energy to make a glucose than we can get out by burning the glucose. I should say, the energy isn't just the ATP. There's also this NADH.

~16:23That NADH that's produced from glycolysis also is storing energy, if you will, from oxidation and an NADH/NAD+ ratio, and you're consuming that going in the opposite direction. You only do gluconeogenesis if you have a high ATP:ADP ratio, of course, and a NADH/NAH+ plus ratio-- that is, if you have it under the right conditions to actually run this pathway and have it make sense for the cell. All right. Now I want to discuss, briefly, how these enzymes in gluconeogenesis work. I'm not going to spend time on fructose 1,6-bisphosphatase or glucose 6-phosphatase. As I said before, this is just hydrolysis of that phosphate-alcohol bond. That's relatively straightforward. But I do want to spend time on pyruvate carboxylase and PEP carboxykinase because this introduces a new reaction, carboxylation. But it also introduces a new concept in metabolism that is actually quite important in eukaryotic cells. And that is this issue of so-called compartmentalized metabolism. And, of course, you've learned in introductory biology that a big difference between eukaryotes and prokaryotes is eukaryotes have all of these organelles-- that is, these membrane-bound structures within cells that carry out various functions. So one reason that it's useful to have these different membrane-bound organelles is it creates different compartments within the cell. What does that mean? Well, if you have different compartments, that means you can create different conditions within each compartment. And that's very important for metabolism. Well, why is that important for metabolism? Well, because, remember, whether or not reactions are favorable depend on delta G, which, of course, is dependent on the equilibrium constant, which is related to delta G 0 prime but also to the ratio of the products and reactants within that compartment. And so by having different compartments, you can have different ratios of metabolites-- say, a different ratio of ATP to ATP or a different ratio of NADH to NAD+. Having those different ratios means that because delta G is proportional to the ratio of the reactants over the products-- I'm sorry-- products over reactants, by having different ratios of things like ATP and ADP, NAD, NADH, you can make different reactions more or less favorable depending on which compartment in the cell that they're located. Now it turns out that glycolysis and most of gluconeogenesis takes place in the cytosol of eukaryotic cells. The cytosol is basically the space inside the cell that's not inside another organelle. However, pyruvate carboxylase is a reaction that takes place in mitochondria because mitochondria have a particularly high ATP:ADP ratio. And it turns out, this helps favor the pyruvate carboxylase reaction. And so we'll talk a lot about reactions in the site cytosol versus the mitochondria. But basically, here's glucose to PEP. Whether you do that in one direction by glycolysis or the other direction by gluconeogenesis, that happens in the cytosol as does turning that PEP into pyruvate. But if you're going to reverse the reaction, eukaryotes take advantage of a different compartment, the mitochondria, with a high ATP to ADP ratio. And basically, in the mitochondria, turn that pyruvate into oxaloacetic acid-- that is, carry out the pyruvate carboxylase reaction. And then that pyruvate carboxylase reaction generates oxaloacetic acid, and then PEPCK can, either in the mitochondria or in the cytosol, generate PEP. And so here's a place where one can run one reaction-- PEP to pyruvate of t make ATP in the cytosol-- but a different reaction-- pyruvate carboxylase to turn pyruvate into oxaloacetic acid that would then be used for PEPCK to turn that back into PEP and have that take place in the mitochondria.

~19:39I want to point out, just so for MCAT exams and things like that, PEPCK is classically defined as a cytosolic activity, at least in terms of gluconeogenesis. Although, there is a mitochondrial isoform of PEPCK. And it's at least somewhat debated. There's some evidence that, at least, in some tissues, that might be important for gluconeogenesis as well. And so that's why I draw it as PEPCK using oxaloacetate to PEP either in the mitochondria or in the cytosol because that's an area of active investigation right now. All right. Let's start by talking about how does pyruvate carboxylase work? This is an example of a carboxylase reaction, that is, adding a CO2 group to a molecule to make a carbon-carbon bond. That purple CO2 is added to the CH3 of pyruvate of to make oxaloacetate. And how this happens, in many cases, uses a cofactor. And that cofactor is called biotin. Biotin, like many cofactors, is a vitamin. And this provides a useful functional group. In this case, enzymes that use biotin use ATP to basically drive CO2 addition to biotin. And then that CO2 on the biotin is then activated to be transferred to another molecule in a carboxylation reaction. All right. So what does biotin look like? I will draw it for you. So this, here, would be biotin. I've drawn it in the enolate form of biotin. I'll draw it in a different way in a second. Biotin is typically bound to a lysine molecule in the active site of an enzyme that uses this. And so this, here, is basically a peptide bond between the terminal amino group, the epsilon amino group of the side chain of a lysine in a residue that basically makes a peptide bond to link biotin into the active site of the enzyme that's using it. Biotin is often drawn in the keto form. And the active part of biotin is basically this top part of the molecule. And so to draw it to see differences between the enolate and keto form, I just want to show that quickly. I drew it in the enolate form because it's easier to see, I think, how it-- so this, here, would be the keto form where biotin is drawn most commonly. And basically, the reaction, the way biotin picks up a CO2 is as follows. And that is CO2 can exist particularly under basic conditions as CO2 or as-- this is bicarbonate. OK. So bicarbonate-- turns out, some enzymes use CO2 directly. Some enzymes use bicarbonate. It turns out that pyruvate carboxylase uses bicarbonate. So biotin bound in the active site of pyruvate carboxylase will utilize bicarbonate in the following way. And I'll just point out that the pH of the mitochondria is also more basic, which also helps this pyruvate carboxylation reaction. All right. So what happens is this bicarbonate is phosphorylated by ATP. This phospho intermediate can then react with this active site here of biotin to release the phosphate. And now, you now have this activated CO2 group that's attached to the biotin. Here's pyruvate drawn in the enol form. You can look back in your notes about how to interconvert it between keto and the enol form. I've shown that several times. And then this, then, can end up adding this CO2 to the end of pyruvate to generate oxaloacetic acid plus regenerate the biotin cofactor in the right form. It'll be the keto form, which can then get back to the enol form. And it's ready to do another catalytic cycle. And so, basically, pyruvate carboxylase uses ATP to produce this carboxylate of biotin. And then uses that to add the CO2 to pyruvate to generate oxaloacetic acid. Now I want to point out that oxaloacetic acid is both an alpha keto acid like pyruvate was. And it's a beta keto acid. And so this ketone is alpha to that carboxylic acid. And it's beta to that carboxylic acid. So it's both an alpha and a beta keto acid. And it turns out that the way PEPCK works is it takes advantage of the favorable decarbonization of a beta keto acid, which we talked about last time, along with GTP to phosphorylate pyruvate and trap it in the enol form.

~23:33And so this is how PEPCK works. So I'm going to draw GTP in this very stylized way-- so guanine with three phosphate groups. There's oxaloacetate. That decarboxylates-- takes advantage of this being a beta keto acid to decarboxylate and add the phosphate. And that's how we can generate PEP. OK. Great. And so that's how you can build a different pathway that uses ATP and GTP for energy input in order to run glycolysis in the opposite direction-- gluconeogenesis. All right. So how is gluconeogenesis regulated? Well, just like the principles we talked about in glycolysis, it works in a way that makes sense. And so you can guess that the steps that are going to be regulated are exactly the ones that you might guess. They were the same steps that were regulated running glycolysis. It's going to be these big changes where energy changes occur across the pathway, which are also the entrance and exit of the pathway. And basically, regulation has to be reciprocal. So in glycolysis, you want to increase glycolysis under conditions where you need to produce ATP. And you want to decrease glycolysis if you have enough ATP or enough of another downstream product, such as citrate. All right. Well, gluconeogenesis is going to be exactly the opposite. You certainly don't want to do gluconeogenesis if you need energy release. If you need ATP, you don't want to run gluconeogenesis. However, you want to increase gluconeogenesis, if the cell has ATP excess, you also want to do it if you have excess of other products like citrate because why go through the trouble of sending things down glycolysis if you have nowhere to put it. You might as well, instead, make glucose or shunt that glucose off to produce glycogen. And so let me just add that regulation here. And so major regulators of gluconeogenesis are as follows. And so high levels of citrate is going to stimulate fructose 1,6-bisphosphatase. So high levels of citrate will inhibit things coming through glycolysis and activate gluconeogenesis by acting at fructose 1,6-bisphosphatase to match the energy considerations. If you have high levels of AMP, energy charge is low. You want to stimulate glycolysis at phosphofructokinase. Similarly, that's going to inhibit gluconeogenesis at FBPase. And it turns out, high levels of ADP-- I'm sorry-- yeah, high levels of ADP, low energy charge, also inhibits PEPCK because you also don't want to try to generate PEP under those conditions. Great. All right. Now in animals, we also want to regulate glucose catabolism and production under signaling control. And this gets back to this other topic we talked about, such as the liver's job being to maintain a constant level of blood sugars. Most of you are very familiar with blood sugar control through diabetes. You've probably heard that this is under hormonal control, that the job of insulin is to lower blood glucose. And you also have hormones like epinephrine or glucagon whose job is to raise blood glucose. Why do you want to raise blood glucose? Well, if you have some kind of fight-or-flight response-- you see that lion, and you need to run away, your body has this adrenaline rush. That's what epinephrine is. That basically wants to supply more energy to your muscles, so that you can run away effectively. So it turns out, both insulin and glucose act on lots of tissues in the body. I'm going to focus on what they do in the liver. And that's because in the liver, this is a major organ that regulates blood glucose levels. So if you have excess glucose around, what do you want to do? You want to stimulate glucose uptake into cells, its metabolism, and its storage. And so insulin with high blood sugar basically wants to stimulate, taking glucose into the liver and storing it as glycogen.

~27:03If you want to run away from the lion, well, now you have epinephrine around. You want to make sure that as you're consuming that glucose in your blood that you're continually making more. And so you want the epinephrine to stimulate release of glucose either from gluconeogenesis or from the breakdown of glycogen. And both of these hormones act-- insulin and epinephrine-- act at a level where they can regulate enzymes of glycolysis and gluconeogenesis as well as entry and exit of glucose monomers in and out of glycogen. And so this is an example of how fructose 2,6-bisphosphate works. And so remember you learned from Professor Yaffe that epinephrine acts via cyclic AMP signaling, which turns on a kinase-- protein kinase A. And that protein kinase A can regulate enzymes that produce or break down, basically, these enzymes that produce or break down fructose 2,6-bisphosphatase which, in turn, regulates PFK activities such that you can match your need to burn that glucose versus doing gluconeogenesis in the case of the liver, so you can do enough gluconeogenesis in order to have glucose around for the body to use in that fight-or-flight response being driven by epinephrine. Now a major effective insulin and epinephrine signaling, though, is actually on release or storage of glucose molecules in glycogen. And I alluded last time in talking about the regulation hexokinase that glucose 6phosphate is basically the entry point to get glucose units in and out of glycogen. And I want to discuss how you add and subtract those glucose units into storage polymers. Now I'm going to discuss it in the context of glycogen storage in humans. But remember, plants actually store things also as glucose polymers. The chemistry is very similar. But, obviously, the regulation is very different.

~28:41Now you'll hopefully recall from past lectures that these storage polymers of glucose-- glycogen in humans, starch in plants-- are basically these alpha 1,4 linkages of glucose molecules. Remember, there was a nonreducing end and a reducing end of the molecule. So starch was a straight-chain polymer. Glycogen had these alpha 1,6 branch points that made this branch polymer, where there's lots of non-reducing ends and a single reducing end. And each of these non-reducing ends is a polymer of glucose molecules with this alpha 1,4 linkage that, at the branch point, has this alpha 1,6 linkage to make this long-chain branched polymer. Now when we discussed this at the time, I mentioned that this is useful because you have all these non-reducing ends that can be used to add or subtract glucose monomers. And that's great. This is a nice compact form of storage with lots of places to put or remove glucose from. You either store it quickly, insulin driving glucose storage-- or remove it quickly, epinephrine driving release of glucose from the glycogen. And so if we're going to have a way to add glucose polymers or break them down, those are two separate pathways, just like glycolysis and gluconeogenesis-- reciprocal activities, but we need two pathways to do it because delta G has to be less than 0 for each pathway to work. Also, we have to be able to regulate these separately because we don't want to have a futile cycle. Now the way you add and subtract glucose polymers to glycogen acts through glucose 6-phosphate being first converted to glucose 1-phosphate. So this, here, is alpha glucose 6-phosphate. Remember, it's alpha because I drew that hydroxyl at the one position pointing down. This, obviously, can enter glycolysis and come in from glucose via hexokinase. All right.

~30:19This is first converted via a mutase reaction to move the phosphate from the 6 to the 1 position of glucose. So this, here, is glucose 1-phosphate. The way the mutase reaction works is exactly the same analogous mechanism that I explained for phosphoglycerate mutase in glycolysis-- moves the phosphate in this case from the 6 to the 1 position of glucose. And then once you have that glucose 1-phosphate, that can be added to a non-reducing end of a glycogen polymer using an enzyme called Glycogen Synthase, GS. So that'll give me a glycogen molecule with one additional monomer added to the non-reducing end of the polymer. And you release that glucose back off via a different pathway, a different enzyme called phosphorylase. All right. Now the activity of these enzymes-- glycogen synthase and phosphorylase-- that is, the pathway to add glucose 1-phosphate glucose monomer to the polymer versus remove it from the polymer is under hormonal signaling control in animals. And basically, that hormonal signaling works as follows. So phosphorylase can be phosphorylated or dephosphorylated via a signaling enzyme, an enzyme like phosphorylase kinase, which is downstream of PKA, which is downstream of epinephrine signaling. OK. And so when phosphorylase is phosphorylated by phosphorylase kinase-- that has, has a phosphate group added is a signaling cascade-- it is in the active state. And when it's dephosphorylated, it's in the inactive state. Well, glycogen synthase also is subject to regulation by protein phosphorylation on the enzyme by a kinase. Except this has the opposite relationship. So when it is in the phosphorylated state, it is inactive. But it is in the non-phosphorylated state, it is active. And so protein kinase A, which is downstream of epinephrine, can both activate phosphorylation of phosphorylase and glycogen synthase. And that makes sense. You want to release glycogen glucose monomers from glycogen. You turn on, by phosphorylation, the pathway to release them. And you turn off the pathway to store them. Another kinase that phosphorylates to glycogen synthase is a kinase called GSK or Glycogen Synthase Kinase. This kinase is inhibited by insulin signaling. And so this is a negative of a negative. So effectively, a negative of a negative will keep it in the inactive state. And so insulin, by inhibiting the ability of GSK to put glycogen synthase in the inactive state, will make glycogen synthase active. And when you have high insulin around, that actives glycogen synthesis, and you store glucose monomers as glycogen. Get them out of the blood. OK. Now let's discuss the chemistry that allows us to do this glycogen synthase and phosphorylates reactions. Now as we go through this, what you will see is that when we break down the polymer, energy is released. So that's going to be breaking down a polymer. That's the right direction of entropy. And so that's going to be released. And storing it is going to require energy. That is, you're going to have to have some energy input to build a polymer. But nature actually does this in a way that is actually quite efficient. First, let's talk about how you store glucose 1-phosphate units by adding them to glycogen. So this uses energy input from UTP, which is an ATP equivalent for exactly the reason I described earlier. That is, because ATP plus UDP, like any other interconversion of nucleoside triphosphates and diphosphates is very close to equilibrium.

~33:26So the UTP to UDP ratio should be similar to the ATP the ADP ratio. And so UTP is really an ATP equivalent. But nature, for whatever reason, decided to use UTP here. And it generates a nucleoside sugar called UDP glucose. And so let me show you what that is. So here is glucose 1-phosphate. I will draw a stylized version of UTP here. And so this molecule with the UDP added to the glucose is UDP glucose. This will also generate a pyrophosphate. And that pyrophosphate can be hydrolyzed to generate two inorganic phosphates, effectively, like building any other polymer, by doing this downstream-- keeping that product of the first reaction low helps pull that reaction forward for polymer synthesis. This UDP glucose can now react with the non-reducing end. So this, here, will be the non-reducing end of the polymer. That's the OH group at the 4 position, at the nonproducing end. And so that then generates that alpha 1,4 bond to add to the polymer. So here, we have glycogen n plus 1 plus releases UDP. And so the net to add a glucose 1-phosphate to the non-reducing end of the growing glycogen polymer is net conversion of a UTP to a UDP. So that is one ATP equivalent to add glucose 1-phosphate to the end of the polymer. Now, of course, you had it to also have an ATP from hexokinase to capture that glucose 6-phosphate to begin with. But effectively, this is two ADP molecules to add a glucose to glycogen. All right. Now if we're generating starch, that's all we have to do, just build a long polymer. But if we're making glycogen, remember, glycogen has all of these branch points on them as well. And to do the branch points, you basically have to make this alpha 1,6 bond, which then gives you two new non-reducing ends that you can add more subunits to. The way nature does this is as follows. And so if I just draw this here in a stylized way, it turns out, once you get to about seven units here in the growing polymer-- so this is the reducing end, and this is the non-reducing end of the polymer. And so the polymer is growing by adding things to that end. Once you get about seven, there's an enzyme that will basically cleave this, and move it and make a new alpha 1,6 bond. OK. Here we go. We have our reducing and our non-reducing end and basically transfers these 7 units over. And now, we have two non-reducing ends that we can continue to grow the polymer from. You can think of this like the airplane analogy. When you load an airplane, at least the way that's most efficient, the people who are sitting in the back of the plane, they get on first. But then, they're the last people off the plane because you add basically to these non-reducing ends, and then you subtract from those non-reducing ends. And so the first glucose added, the one at the reducing end, is the last one that is going to be removed. All right. Now phosphorylase is the enzyme that allows you to do the opposite-- that is, to break that alpha 1,4 bond and release monomers. And it's called phosphorylase because it uses phosphate to break that alpha 1,4 bond, taking advantage of the fact that breaking down the polymer is favorable and actually releasing the glucose from it again is a glucose 1-phosphate. And so at the non-reducing end of the molecule-- so this, here, would be the non-reducing end of the glycogen polymer. You basically have a phosphate molecule that takes that off, so you have a glycogen n minus 1 plus a glucose 1 phosphate, which can now undergo a mutase reaction back to glucose 6-phosphate that could then be released from the cell if it's a liver, or it could be burned in glycolysis.

~37:02Now, obviously, if you're doing breakdown, that is, your chewing back from non-reducing ends, eventually, you're going to hit a situation where you come to where there's just a nub here with an alpha 1,6 bond there. That is, chew it back till you hit this single monomer with a non-reducing end here to this alpha 1,6 break point. Turns out, you just cleave off that nub, if you will. And so that nub is then just released as free glucose. So other than release of that nub, what it means is that you actually basically get no ATP to actually break down in the phosphorylase reaction. So the net cost to store a glucose 1-phosphate molecule is one ATP via the UTP to UDP conversion in the glycogen synthase reaction. And you get that one glucose 1-phosphate back. And so two ATPs to put a glucose tin glycogen. And then you get it directly back out as a glucose 1-phosphate. That's a phosphorylated glucose that can then enter glycolysis. So that's one less phosphate you need to spend on glycolysis. And so it's incredibly efficient to store glucose in that way. And so it's actually slightly greater than one ATP to store it as glycogen because you lose in ATP with that nub removal at the branch polymer. But for all intents and purposes, it costs one ATP to store your glucose as glycogen, which is pretty amazing. All right. Now before I leave the topic of sugar storage, I want to make some comments on some sugars that are stored in forms other than glucose. And want to talk about these because these sugars are, of course, important parts of our diet. And so as we've mentioned in prior lectures, two of these sugars are sucrose and lactose. So remember, sucrose is a disaccharide of glucose plus fructose. And lactose is a disaccharide of glucose plus galactose. So those are storage forms of sugar-- sucrose in the case of plants, lactose in the case of milk made by mammals. And so to break those sugars down, basically, you break the disaccharide. Now, you have the glucose molecules you know how to handle. That's glycolysis. But I want to spend a little bit of time how you deal with the fructose and the galactose that you get out from these sugars because this actually relates to some things that I'm sure you've all read about in the news. For instance, fructose as a sugar in our diet is actually quite controversial. Lots of stuff out there that says fructose was cooked up in the devil's kitchen and is this evil thing. There's actually others who argue that, oh, fructose is the same as glucose, the same number of calories. There's both sides of the debate. Both sides are actually making factual claims. And I want you to understand the biochemistry, so you can actually judge for yourself who's right in those claims. All right. So let's start with talking about how you metabolize fructose. And so fructose is first captured by phosphorylation to generate fructose 1-phosphate. This is carried out by an enzyme called ketohexokinase. Ketohexokinase is an enzyme this present in your gut and your liver. And so fructose in the diet, from breaking down sucrose or from high-fructose corn syrup, whatever, is captured with ketohexokinase to make fructose 1-phosphate. Turns out that phosphofructokinase, our old friend from glycolysis, is less efficient than ketohexokinase but can also carry out this reaction. And remember, what is it do in glycolysis? It adds a phosphate to the 1 position of fructose 6phosphate. Well, it can also add a phosphate to the 1 position of fructose give you fructose 1-phosphate.

~40:24OK. Once you have this fructose 1-phosphate, which, remember, is not in glycolysis, it can be a substrate for the glycolytic enzyme aldolase. What does aldolase do in glycolysis? It splits FBP into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Well, here, you only have a phosphate on the 1 position. And so if you look back at how aldolase works, what you'll see is that if aldolase acts on fructose 1-phosphate, you'll generate a dihydroxyacetone phosphate. That phosphate will go to the dihydroxyacetone half, which, of course, is perfectly good substrate for glycolysis. The other product of aldolase acting on fructose 1-phosphate is just glyceraldehyde without the phosphate on it. And then there's another enzyme that will phosphorylate glyceraldehyde to generate glyceraldehyde 3phosphate, which can then also, of course, enter glycolysis. And so fructose is a hexose just like glucose. You generate two trioses-- DHAP and glyceraldehyde 3-phosphate. That costs you two ATP to do it, exactly the same cost is what you spent to get glucose to make DHAP and glyceraldehyde 3-phosphate. And so people are absolutely right when they say metabolizing fructose has exactly the same caloric cost as metabolizing glucose-- two ATPs to get it into the trioses. And then whatever you get out of it, you get out of it two ATPs, four ATPs-- so net two ATPs if you just do fermentation, more if you do complete oxidation. But the bottom line is that from that standpoint, you get exactly the same number of calories from burning glucose versus fructose. However, I want to point out that the regulation will not be the same. And that's because in this fructose metabolism, you will notice that there was no glucose 6-phosphate and no FBP generated. That means you are avoiding all of the feedbacks that control how much glucose you send to glycolysis-- first to glycogen. Remember, that's what glucose 6-phosphate does, acting on trapping sugar. And more importantly, you're actually not using phosphofructokinase in a way to fit with the rest of the feedback regulation. I also show that here on this slide. It's basically a better version of what I drew up here. And I now want to point out this storage of citrate and this feedback regulation of citrate. So we'll talk about the specifics of this more in other lectures. But it turns out, citrate is the precursor for how one stores carbon as fat. And so if you're putting fructose glucose into the system and citrate is too high, it'll shut off phosphofructokinase and stop carbon coming down the system, shunting it instead to glycogen or just saying don't give me any more glucose at all. If I'm doing the same thing with fructose, those feedbacks don't exist. Citrate doesn't feed back on any of the enzymes that are regulated for fructose metabolism. And so the idea is carbon keeps getting dumped into citrate, which ultimately ends up as fat. And this really underlies, at least on a theoretical basis, why there may be a connection between fructose and obesity or metabolic syndrome. I've posted articles on Stellar, one from The Popular Press, one from a scientific journal that discusses the controversy, discusses this. But now, at least, you have the background to draw some of your own conclusions. OK. That's fructose metabolism. What about galactose metabolism? Well, galactose metabolism interfaces with, effectively, glycogen metabolism or, at least, glucose 1-phosphate-or sorry, UDP glucose and does so in a way that is a little bit non-intuitive. But it's actually very important to understand galactose metabolism, at least for those of you who are looking to go to medical school.

~43:42It turns out that galactose metabolism and issues with metabolizing galactose are what underlie a set of rare genetic diseases leading to a set of conditions called galactosemias. I guess, if you're a pediatrician, you may encounter these. If you're not, these are quite rare. Although med schools love to ask about these, and certainly, they're very popular on things like board exams. So it's something you should know about. And so I just want to briefly introduce galactose metabolism to you.

~44:09So this is galactose. Like all sugars, galactose is initially trapped by phosphorylation. It's phosphorylated on the 1 position to trap it, just like fructose. And so this is galactose 1-phosphate. Remember, galactose is an epimer of glucose. So it differs from glucose here by the stereochemistry of the hydroxyl group at this 4 carbon. And so the way galactose, once it's trapped as galactose 1-phosphate is metabolized is it will react with UDP glucose. So here is UDP glucose from glycogen metabolism drawn in a very stylized way. That will generate a UDP galactose and a glucose 1-phosphate, which, of course, can enter glycolysis, can be stored as glycogen, et cetera. This UDP galactose can then be turned into UDP glucose via an epimerase reaction. And so remember, the epimerase reaction needs to change the stereochemistry of the hydroxyl group at this 4 position, such that it's pointing up in galactose or pointing down in glucose. The way epimerases work is that they require a cofactor. That cofactor is NAD. and they work in the following way. And so if I just draw, here's the 4 position of galactose, the 4 carbon of galactose, with the hydroxyl group pointing up, an epimerase can basically remove a hydride from the bottom face of the molecule, transfer those two electrons to NAD to generate NADH. Now, you get this lactone intermediate at the 4 carbon. And then you can have two electrons from an NADH can be added to the opposite face of the molecule that regenerates an NAD+. And now, we've effectively changed the stereochemistry at the 4 carbon to now be pointing such the hydroxyl points down, and that's glucose. And so the way epimerases work is they basically remove and add electrons from different faces of the pyranose ring. And that changes the stereochemistry of which direction the hydroxyl group is pointing. And so effectively, this is how we metabolize all kinds of different sugars, either putting them in glycogen or other starch, some other storage carbohydrate that we can get them later, and then use them in glycolysis as a way to drive glucose oxidation as a way to release energy and keep ATP high in the cell. All right. Now I want to shift topics and begin to discuss how we can oxidize pyruvate from glycolysis to make CO2 as an alternative to how we can release energy from carbohydrate oxidation rather than just use fermentation. Now you'll see that the enzymes and ways that we do this is going to also be how we make citrate. And so this isn't just about releasing energy. It's also about storing energy because you'll see, citrate is a precursor for storage as fat. The reactions downstream of pyruvate oxidation also make lots of other useful intermediates that can be used to synthesize other things cells need-- amino acids, nucleic acids. And so this pathways we're going to discuss are going to come up over and over again throughout the course. Now to facilitate this discussion, I'm really going to at least initially focus on catabolism.

~46:58And so just to remind you, if we do glycolysis by turning glucose pyruvate, this involves oxidation. So we need to dispose of those electrons somewhere. In other words, that NADH has to be regenerated NAD. Fermentation of pyruvate into something like lactate is a way to do that and allows net ATP production from glycolysis without the need for any oxygen. However, if we're going to completely oxidize that pyruvate CO2 we're going to generate many more electrons as waste that we have to put somewhere. Oxygen is a particularly good electron acceptor. And so we can reduce oxygen to water as a way to deal with that electron waste, and that allows us to, just burning wood, release lots of energy from glucose by transferring those electrons ultimately to oxygen. And of course, that same process-oxygen to water-- if we're using the pyruvate to fully oxidize it, also is necessary to regenerate the NAD to run glycolysis. Now ultimately, the series of reactions that allow chemically conversion of pyruvate to CO2 is called the TCA cycle for try Tricarboxylic Acid Cycle or sometimes referred to as the Krebs cycle and also is referred to as the citric acid cycle-- so TCA, Tricarboxylic Acid Cycle, Krebs cycle, citric acid cycle-- all synonyms for the same thing. What these cycles are is basically a way to take two carbon units. And those two carbon units can come from the oxidation of pyruvate or from the oxidation of lots of other fuels. And those two carbon units are combined with four carbon units. You'll see oxaloacetate is the four-carbon unit-- oxaloacetate gluconeogenesis. That oxaloacetate could come from the pyruvate-carboxylase reaction. Or it could come, as you'll see, from the TCA cycle itself. This generates a six-carbon molecule, which is citrate. Citrate is a tricarboxylic acid, hence the citric acid cycle or the tricarboxylic acid cycle. And then those six carbon units are reoxidized, releasing two CO2 molecules, back to four-carbon oxaloacetate. And hence, this is a cycle. Now this cycle was initially described by Hans Krebs, who predicted the cycle largely based on chemistry-- one of the most amazing feats in the discovery of metabolism. Turned out, he was right. And thus, it's also referred to as his name, the Krebs cycle. Now lots of oxidation is happening in this cycle. You're oxidizing the molecule, releasing CO2. That means lots of energy is released. Those electrons ultimately will be transferred oxygen. And so we can use this to make lots of ATP and do lots of other work. Where are these reactions all occur is in the mitochondria, specifically in the matrix of the mitochondria. Remember, the mitochondria, from cell biology, is a double-membrane organelle. The innermost part of the mitochondria is referred to as the matrix. And these have conditions that favor pyruvate carboxylase reaction, as I talked about, but also favor other oxidation reactions, such as those that occur in the TCA cycle. And so next time, what I will start with is by being more explicit about what chemistry is happening to allow these two carbon units to combine with oxaloacetate 4-carbon to generate citrate and go over the reactions that then allow you to run a cycle to regenerate oxaloacetate, that is, run the TCA cycle as a way to oxidize carbon. Thanks.

3.3丙酮酸脱氢酶复合体（PDH Complex）：三酶五辅酶的分子摆臂接力与门户调控

一、丙酮酸跨膜入线粒体与辅酶 A（CoA-SH）的活化本质

当细胞氧气充足时，糖酵解在细胞质中生成的 2 分子丙酮酸不会去走发酵的“权宜之计”，而是要进入线粒体基质（Mitochondrial matrix）（原核生物则在细胞质）被彻底氧化为 CO₂ 和 H_2O。然而，线粒体拥有双层膜结构：丙酮酸可以自由穿过外膜的孔蛋白（Porin/VDAC），却无法直接穿透致密的线粒体内膜，必须借助内膜上的专一转运蛋白——线粒体丙酮酸载体（MPC，又称丙酮酸移位酶，以 H⁺/丙酮酸 同向转运方式将丙酮酸运入线粒体基质）。

在 L23 黑板上，Vander Heiden 教授特意画出了辅酶 A（CoA-SH）、FAD 和硫辛酸的完整化学结构，帮我们撕掉了缩写符号带来的错觉：

1. 辅酶 A（Coenzyme A, CoA-SH）：平时我们简写为 -S-CoA，好像只是个小小的含硫尾巴，但它实际上是由 3'-磷酸腺苷二磷酸（3'-phospho-ADP） + 泛酸（Pantothenic acid，即维生素 B₅） + β-巯基乙胺（β-mercaptoethylamine）三部分拼成的巨型分子！最末端 β-巯基乙胺上的游离巯基（-SH）是它的活性功能基团，能与羧酸形成高能硫酯键（Thioester bond，如乙酰-CoA 的 CH_3CO～S-CoA，水解 Δ G'^° = -31.4 kJ/mol）：由于硫原子的 3p 轨道与羰基碳的 2p 轨道重叠极差、缺乏普通氧酯的共振稳定化，既使羰基碳具有极强的亲电性（优秀酰基供体），又使其 α-甲基氢易去质子化形成碳负离子！
2. 黄素腺嘌呤二核苷酸（FAD）：衍生自维生素 B₂（核黄素，Riboflavin）。虽然名字叫“二核苷酸”，但它的黄素一端连接的并不是环状核糖，而是五碳开链多元醇——核糖醇（Ribitol）！其核心活性部位是异咯嗪环（Isoalloxazine ring）：氧化态 FAD 在异咯嗪环中央拥有共轭双键体系，因此呈现鲜艳的黄色（拉丁文 flavin 即黄色）；当它接受 2 个电子和 2 个质子（通常以 1 个氢负离子 H^- 加 1 个 H⁺，或分步接受单电子）还原为 FADH₂ 时，中央共轭双键消失，溶液随之变为无色！
3. 硫辛酸（Lipoic acid）：一种含八碳脂肪酸骨架并在 C6 和 C8 位连有两个硫原子的水溶性维生素样辅因子。它没有英文缩写，通过羧基与 E2 亚基赖氨酸残基的 ε-NH₂ 形成酰胺键，构成长约 1.4 nm 的柔性摆臂——硫辛酰胺（Lipoamide）。其末端的五元环二硫键（-S-S-，氧化态）与二氢硫辛酰胺（含两个游离巯基 -SH，-SH，还原态）之间可以可逆地互变，从而承担起“既搬运二碳乙酰基、又传递一对电子”的双重重任！

二、丙酮酸脱氢酶复合体（PDH Complex）：三酶五辅酶的四步链式催化

进入线粒体基质后，丙酮酸遇到了连接糖酵解与柠檬酸循环的总枢纽——丙酮酸脱氢酶复合体（Pyruvate Dehydrogenase Complex, PDH 或 PDC）。它催化如下不可逆的氧化脱羧反应：

丙酮酸 + CoA-SH + NAD⁺ ──(PDH 复合体（3 酶 5 辅酶）)──► 乙酰-CoA + CO₂↑ + NADH + H⁺ (Δ G'^° = -33.4 kJ/mol)

丙酮酸氧化脱羧是葡萄糖有氧氧化过程中第一个产生 CO₂ 的反应，位于线粒体基质（真核），反应严格不可逆 2012·填空。这一反应之所以无比精巧，是因为它要把脱羧（断裂 C–C 键）、氧化（将醛基氧化态提升至羧酸氧化态并捕获电子）和高能硫酯键合成（接入 CoA-SH）三件大事一气呵成！为此，自然界将 3 种酶（E1 丙酮酸脱氢酶、E2 二氢硫辛酰转乙酰酶、E3 二氢硫辛酰脱氢酶）和 5 种辅酶/辅基（TPP、硫辛酸、CoA-SH、FAD、NAD⁺） 2009/2014·单选 以非共价键精密组装成了直径达 30～ 50 nm（比核糖体还大！）的巨型多酶复合体（Multienzyme complex） 2009·名解。

3D3D 分子：E1 丙酮酸脱氢酶组分（TPP 催化脱羧生成羟乙基-TPP）步骤 1：TPP C2 碳负离子亲核加成丙酮酸，释放第一分子 CO₂↗

让我们沿着硫辛酰胺摆臂的转动轨迹，拆解 PDH 复合体内部环环相扣的 5 步接力机制：

1. 第 1 步（E1 催化 α-脱羧）：丙酮酸 → 羟乙基-TPP + CO₂↑
   E1（丙酮酸脱氢酶组分，Pyruvate dehydrogenase）的活性中心结合着辅基 TPP（焦磷酸硫胺素，维生素 B₁ 的活性形式） 2009/2013·单选/名解。正如我们在第二章乙醇发酵中学过的，TPP 噻唑环 C2 位极易解离质子形成亲核性的碳负离子（叶立德 ylide），进攻丙酮酸 α-酮羰基碳并促发 α-脱羧释放 CO₂，随后利用带正电的噻唑环季氮原子作为“电子槽（electron sink）”共振稳定脱羧后的负电荷，生成共价结合在 E1 上的羟乙基-TPP（Hydroxyethyl-TPP）中间物。

• Vander Heiden 教授对比点拨：在酵母乙醇发酵的丙酮酸脱羧酶中，羟乙基-TPP 直接质子化就释放出了乙醛（非氧化脱羧，不产 NADH）；但在有氧氧化的 PDH 复合体中，羟乙基-TPP 立即被交给 E2 的氧化态硫辛酰胺进行氧化脱羧并生成高能硫酯键！

1. 第 2 步（E1 与 E2 协同催化氧化还原与转乙酰基）：羟乙基-TPP + 氧化态硫辛酰胺 → 乙酰二氢硫辛酰胺 + TPP
   E2（二氢硫辛酰转乙酰酶，Dihydrolipoyl transacetylase）伸出长长的氧化态硫辛酰胺（含二硫键 -S-S-）摆臂，探入 E1 的活性口袋。羟乙基-TPP 的碳负离子亲核进攻硫辛酰胺的二硫键，在这一瞬间同时完成了两件事：① 羟乙基被氧化成了乙酰基（-COCH₃），而硫辛酰胺的二硫键接受电子被还原打开；② 乙酰基通过高能硫酯键共价挂在了硫辛酰胺的 C8 巯基上，生成乙酰二氢硫辛酰胺（Acetyl-dihydrolipoamide），同时再生出自由的 E1-TPP！

1. 第 3 步（E2 催化酯交换生成乙酰-CoA）：乙酰二氢硫辛酰胺 + CoA-SH → 乙酰-CoA + 二氢硫辛酰胺
   挂着乙酰基的硫辛酰胺长臂从 E1 摆回 E2 自身的催化中心，游离的辅酶 A（CoA-SH）进入，其巯基亲核进攻乙酰二氢硫辛酰胺的羰基碳，发生同能级的硫酯键交换反应，释放出产物 乙酰-CoA（Acetyl-CoA）！此时，E2 的摆臂变成了带有两个还原态巯基（-SH）的二氢硫辛酰胺（Dihydrolipoamide）。

1. 第 4 步（E3 催化摆臂再氧化）：二氢硫辛酰胺 + E3-FAD → 氧化态硫辛酰胺 + E3-FADH₂
   如果二氢硫辛酰胺一直停留在还原态（-SH，-SH），E2 就无法去接下一轮的羟乙基！于是，摆臂继续摆向 E3（二氢硫辛酰脱氢酶，Dihydrolipoyl dehydrogenase）的活性中心。E3 上的辅基 FAD 将二氢硫辛酰胺的两个巯基重新氧化回环状二硫键（氧化态硫辛酰胺，满血复活准备下一轮循环！），而 FAD 自身则接受 2e^- + 2H⁺ 被还原为与酶紧结合的 E3-FADH₂。

1. 第 5 步（E3 催化电子终极交接）：E3-FADH₂ + NAD⁺ → E3-FAD + NADH + H⁺
   最后，紧结合在 E3 上的 FADH₂ 将氢负离子转移给游离的辅酶 NAD⁺，生成可扩散的 NADH + H⁺ 进入线粒体呼吸链（每分子线粒体 NADH 氧化磷酸化产生 2.5 ATP），同时使 E3-FAD 恢复氧化态！

三、PDH 复合体的双重严密调控：别构反馈与共价修饰

因为 PDH 反应一旦把三碳丙酮酸脱羧变成二碳乙酰-CoA，在动物体内就永远回不到葡萄糖了（不可逆地损失了生糖潜力！），所以细胞对这道“单向旋转门”实施了极其严苛的双重调控：

1. 产物反馈与能荷别构调控：

• 别构抑制剂（高能/底物过剩信号）：产物乙酰-CoA 直接反馈抑制 E2，产物 NADH 直接反馈抑制 E3；高浓度 ATP 和长链脂酰-CoA 也别构抑制该复合体。
• 别构激活剂（缺能/底物充足信号）：AMP、ADP、游离 CoA-SH 和 NAD⁺ 别构激活该复合体。

1. 共价修饰调控（针对 E1 亚基的磷酸化/去磷酸化）：
   在真核生物 PDH 复合体上，还紧密结合着两个专门的调节酶——PDH 激酶（PDK）和 PDH 磷酸酶（PDP）：

• 磷酸化 → 失活：当线粒体内能荷极高，或正在大量进行脂肪酸 β-氧化产生高浓度 ATP、乙酰-CoA 和 NADH 时，这三者会强力激活 PDH 激酶（PDK），PDH 激酶催化 E1 亚基上的特定丝氨酸残基发生磷酸化，使整个 PDH 复合体失活（从而在饥饿或高脂饮食时关闭丙酮酸氧化，保住宝贵的丙酮酸去走糖异生！）。相反，高浓度底物丙酮酸和 ADP 则强烈抑制 PDK，维持 PDH 活性。
• 去磷酸化 → 激活：当肌肉剧烈收缩导致线粒体 Ca²⁺ 浓度升高，或餐后高血糖刺激胰岛素（Insulin）分泌时，Ca²⁺ 和胰岛素可激活 PDH 磷酸酶（PDP），切除 E1 上的磷酸基团使其去磷酸化而恢复活性，加速丙酮酸转化为乙酰-CoA（用于肌肉氧化供能或脂肪组织合成脂肪酸）！

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🎧 MIT 7.05 课堂原声英文实录对照（本节关联 2 讲 · 完整逐段实录）点击展开 / 收起 ▾

🎬 MIT 7.05 Lec 22 · Gluconeogenesis, Glycogen & TCA Cycle I⏱ ~50:00

~00:00MITOCW | 22. Glucogenesis/Carbohydrate Storage/TCA Cycle I

~00:02PROFESSOR:

~00:02So last time, we discussed glycolysis as a pathway. And I just want to remind everybody that, like all pathways, glycolysis has to be favorable. That is, delta G across the entire pathway has to be less than 0. We spent a lot of time discussing the regulation of glycolysis. And I just want to revisit this quickly. And remember, we discussed that the steps that are regulated are those with the biggest, quote, unquote, bio-energetic cost. That is, the biggest drop in delta G is as molecules would proceed across the pathway. And those are shown here-- the hexokinase step, the phosphofructokinase step, and the pyruvate kinase step of the pathway. Now this is largely true of most pathways that we will consider, in that these regulated steps are often those with the biggest bio-energetic cost. And the reason for that is that it makes a lot of sense. Once you go through those steps, it's very difficult to go back.

~00:55Also, you'll note that these are the steps that regulate entrance and exit from the pathway. And this also is something we'll commonly see and other pathways and also makes some sense. Just to remind everybody, I drew up here a skeletonized version of glycolysis and the most important regulatory features of it. Just to remind people that initially, hexokinase is regulated by glucose-6 phosphate. Glucose-6 phosphate negatively regulates hexokinase. And that's because if the glucose-6 phosphate levels in the cell go up such that it can't be shunted to glycogen-- something we'll talk about today-- or be sent through glycolysis, it makes sense not to trap more glucose. Once glucose-6 phosphate enters the rest of the glycolytic pathway, the key step of regulation is phosphofructokinase. It, of course, will have positive feedback, saying, let's put carbon into the pathway. If energy charge low, AMP is high, activates it. This molecule fructose 2,6-bisphosphate that we talked about earlier today-- we'll mention again today-- can be involved in some other signaling inputs to allosterically regulate the enzyme. FBP can feed forward to activate pyruvate kinase. You want to match input with output. And, of course, if PEP builds up, you want to slow it down. If a downstream product-- citrate-- builds up, you want to slow it down. And of course, a major output of glycolysis is ATP. And if ATP is high, no reason to continue oxidizing glucose carbons. Now this regulation we discussed is often done at the allosteric level by controlling enzyme rates. That can be via a small molecule binding, as I've shown up here. And it could also be via signaling. And that's really a place where fructose 2,6-bisphosphate as a combination of signaling, and allosteric small molecules comes into play. That is, you can have signaling that controls the enzymes that generate fructose 2,6-bisphosphate, thereby creating an additional molecule that can act as allosteric regulator of the enzyme PFK to generate FBP and commit carbon to glycolysis.

~02:44Now we closed the discussion last time beginning to discuss how it is that organisms can make glucose. Obviously, this glucose carbon that is being oxidized to get ATP has to come from somewhere. We mentioned that plants make glucose during the day when they have plenty of excess energy from light from the sun but then use that glucose to do constant metabolism to maintain their ATP levels at night. And we as animals obviously take advantage of this by eating the glucose that ultimately was created by plants using the energy of the sun. We began to discuss an example of where us, as organisms, also make glucose. Of course, this is not net production of glucose. But for instance, if our muscle cells are working hard, lactate can build up. That lactate is excreted into the blood. That lactate can then be used by the liver to regenerate glucose. And one of the major jobs of the liver is to maintain a constant level of blood glucose to continue to supply fuel for various organs, including the muscle to be able to carry out the work that it needs to do. And so this whole process is called the Cori cycle. And a major job of the liver is thus generating glucose to maintain blood glucose levels. And the pathways used to do this is a pathway that we began to talk about last time called gluconeogenesis. And it's really a discussion of gluconeogenesis. That is, how can one build a pathway in plants, in the liver, wherever, as a way to generate glucose from something like lactate? Now, of course, we know from last time, lactate dehydrogenase can, obviously, it's a redox reaction to interconvert with pyruvate. And what gluconeogenesis really is it's a pathway that cells use to convert pyruvate or really anything else that can come into glycolysis-- and example being lactate. This, of course, can happen by the lactate dehydrogenase reaction. And then once it has that pyruvate, turn that pyruvate into glucose. That's really what we're talking about when we're discussing gluconeogenesis. Now how can we turn pyruvate into glucose? Well, we just discussed the whole pathway where we could turn glucose into pyruvate-- glycolysis. And so this is really the reverse of glycolysis. I said many times that all enzymes are reversible. So why can't we just run the pathway in the opposite direction? Well, hopefully, if you've been paying attention for the last several lectures, you realize there's two problems with doing that. The first is that, remember, why we're doing metabolism in the first place is to maintain a high ATP:ADP ratio under conditions so that the cell can use that ATP:ADP ratio to carry out otherwise unfavorable processes. And so thus, at a very high level, cells can't stop catabolism. Glycolysis or whatever has to continue to maintain ATP in the right range. And so now, if we're going to do something anabolic-- that is, build something, turn it into glucose, this has to happen in the background where catabolism has still happened. And so at least you need some kind of way to control both and prevent this from being some kind of futile cycle. Second, even if that ATP level is super high in the cell, it has extra energy, there is still a thermodynamic problem with just reversing glycolysis. And that is, remember, delta G has to be less than 0 for any pathway to work. And so it then stands to reason that if glucose oxidation to pyruvate, if this reaction is favored, as it is in glycolysis, that delta G is less than 0.

~06:03Well, under the conditions where that's occurring, if we want to then say, well, what about the reverse? Let's switch the signs-- pyruvate to glucose. We know that delta G is the same as it was in the opposite direction but, now, changing the signs from positive to negative or negative positive. And so, thus, delta G, for the reverse reaction, must be greater than 0 and, therefore, must be spontaneous-- is not spontaneous. And therefore says that we need to have a different pathway simply for energetic reasons if we're going to go in the opposite direction. Now oxidizing glucose releases energy. That's how we keep the ATP:ADP ratio high. It stands to reason then that we are going to have to put energy in if we're going to run in the opposite direction. And so if we're going to build a pathway from pyruvate to glucose, you can guess, we're going to have to add couple that pathway to ATP-ADP hydrolysis-- that is, couple it to something favorable to now allow this otherwise unfavorable reaction to happen. Now this energy, of course, in plants can come from the sun. We'll discuss that in great detail when we get to photosynthesis. However, for the sake of you and I, I guess, what it says is, if you think about this, if you release energy from glycolysis, it makes sense then you have to add energy if you're going to do gluconeogenesis. But remember, to satisfy all the laws of thermodynamics, the net energy loss to the universe is going to be-- must exist, which means that it is going to cost more energy to make glucose than you're going to get back from breaking it down. That just, hopefully, fits with your intuition. And it actually, I guess, is good news, because when are we going to do anabolism? When are we going to store all these excess calories as glucose? Well, we're going to do it if we overeat. And if we overeat, and it costs us energy in the form of calories to actually store that energy for later, I guess, it means that when we overeat, we don't have things quite as bad as it could have been. All right. Now let's get back to the how one can actually do this. So if we want to think, what is a pathway that we can build to get from pyruvate back to glucose? Well, we know we're going to have to burn some ATP to do it. But what are the issues with simply reversing glycolysis? Well, we can come back here and say, well, what was driving glycolysis in the first place? And it was these big steps of free energy change acts of hexokinase, phosphofructokinase, and pyruvate kinase. Where as many of these other reactions, at least under these standard conditions that are estimated to exist in cells, are relatively close to equilibrium. So there's really no problem with reversing glycolysis across here or across there. The real issue comes from getting around the hexokinase to phosphofructokinase and the pyruvate kinase step. These are the places where the most energy is released, if you will, when you're going through glycolysis. So those are the steps we're going to have to add energy in order to reverse build a pathway that goes in the opposite direction and allows you to start with pyruvate and end up with glucose. And so what we will find is that gluconeogenesis and glycolysis use many of the same enzymes. However, not all of the enzymes are the same because we need different enzymes if we're going to get around this hexokinase, phosphofructokinase, and pyruvate kinase steps of the pathway. And so for gluconeogenesis, we essentially need four new enzymes in order to get past those places. So we're going to need a new enzyme called glucose 6phosphatase. And glucose 6-phosphatase is going to allow us to get from glucose 6-phosphate and turn it back into glucose.

~09:41All right. We're going to need an enzyme fructose 1,6-bisphosphatase, which is going to allow us to take FBP and turn it back into fructose 6-phosphate. And then we're going to need two enzymes to get around the pyruvate kinase step. These are two enzymes called PEPCK and PC. That stands for phosphoenolpyruvate carboxykinase and pyruvate carboxylase. I'll discuss these in great detail in a minute. And these are going to allow us to get from pyruvate back to PEP. Also, essentially, I'll add them up here to this diagram. So to get back from pyruvate to PEP, two enzymes-pyruvate carboxylase and phosphoenolpyruvate carboxykinase to get from fructose 1,6-bisphosphate, fructose 6phosphate, an enzyme called FBPase. And to get from glucose 6-phosphate back to glucose, an enzyme called glucose 6-phosphatase. OK. Now, of course, if you think back, we're also going to need to balance electrons in the pathway. Remember, glucose to pyruvate is an oxidation reaction. That is, we're generating NADH. That means to go in the reverse direction, we need a source of NADH to reverse that GAPDH reaction. I just want to point out that you're only doing gluconeogenesis if cells have plenty of energy. What does plenty of energy mean? Well, that means the ATP:ADP ratio is high because you're doing lots of metabolism. But if you're doing lots of metabolism, and the ATP:ADP ratio's high, that also means the NADH/NAD+ ratio is high. And so you can think of the NAD regeneration by gluconeogenesis as being, for all intents and purposes, an alternative to fermentation, if you will, as a way to keep catabolism going. That is, you're only doing this if you have plenty of NADH around. And so dealing with that electron balance is less of an issue. OK. Now let's discuss briefly how some of these enzymes work. So glucose 6-phosphate and fructose 1,6bisphosphatase are very straightforward. Effectively, all they are is simply removing a phosphate. That's very favorable. Remember, when we did those reactions of hexokinase phosphofructokinase reactions and glycolysis, that, we had to couple otherwise unfavorable things-- adding a phosphate group to ATP hydrolysis. This is the reverse of that. And so the reverse of that, delta G is going to be less than 0. Delta G less than 0 means we just have a phosphatase there, just like you might have on a kinase enzyme. That's very straightforward. But there's a much bigger issue to get around this other step, this pyruvate kinase step. Remember, equilibrium strongly favored PEP to pyruvate conversion. That was one of the reasons why we could use that step to maintain a high ATP:ADP ratio in cells. Well, now, the issue becomes, how can we trap that phosphate back onto pyruvate while generating phosphoenolpyruvate? How one does that is an issue. Even if the ATP:ADP ratio is high, it's never high enough to reverse pyruvate kinase on its own. And so we need a new pathway with two steps in it. That's why there's two enzymes-- PC and PEPCK-- that are there to create a pathway where pyruvate to PEP now becomes favorable. That is, where delta G 0 prime is less than 0. That is, the equilibrium favors to the right. And under cellular conditions, this pathway can actually happen such that cells can net make glucose. Now these are two steps, both of which require ATP. And so you effectively get one ATP from PEP to pyruvate conversion. It costs you to ADPs or two ADP equivalents to go back from pyruvate to PEP, which makes sense. You should have to spend more energy to go upstream than you get back coming downstream. All right. Here's how this pathway works.

~13:03So this is pyruvate, the first reaction catalyzed by PC, which stands for pyruvate carboxylase. Couples ATP hydrolysis to addition of a CO2 that generates this intermediate, which is called oxaloacetate, often abbreviated OAA for Oxaloacetic Acid. That oxaloacetate is then decarboxylated. That is, the CO2 that is lost is removed-- that was added is removed. This reaction is coupled to GTP and is catalyzed by this enzyme, PEPCK PEP carboxykinase and uses that phosphate from GTP to trap pyruvate in the enol form as PEP or phosphoenolpyruvate. great. So once you've regenerated PEP, now that PEP gets to this flat part, if we go over here to our delta G place. So now it can easily run back across this flat part of the curve. That is, go through enolase phosphoglycerate mutase, phosphoglycerate kinase, GAPDH, taking advantage of the high NADH/NAD+ ratio that would exist when you're doing gluconeogenesis-- triosephosphate isomerase, aldolase. This generates a DHAP and a glyceraldehyde 3-phosphate that you can then combine to give a fructose 1,6bisphosphate. Now we get back here to now we have this steep part there. But rather than going through FBP, we can just have a phosphatase, fructose 1,6-bisphosphatase that releases the phosphate. Now, we have fructose 6phosphate. That fructose 6-phosphate can then go through glucose 6-phosphate isomerase and generate glucose 6phosphate. That now gets back to this other steep hill. So this is relatively flat. Now, you have this steep hill around hexokinase. But rather than coupling it to adding to ATP-ADP hydrolysis, you just remove the phosphate with glucose 6-phosphatase. And in the end, now we have a pathway using these four extra enzymes that give us a way to start at pyruvate and run the opposite direction and generate glucose. And you can do so in a way that is thermodynamically favorable. In other words, we can start with 2 pyruvate and use 4 ATP-- so and ATP to turn pyruvate twice into oxaloacetate. So that's two. And then another ATP at phosphoglycerate kinase to go backwards there to go from-- to run that reaction backwards, plus 2 GTP. Those 2 GTP are effectively the equivalent of an ATP. So this would be 6 ATP equivalents. Why are the GTPs equivalent of ATP? I just want to point out that cells have enzymes that basically can interconvert ATP and GDP or, really, any nucleoside triphosphate and diphosphate to GTP plus ADP. This has a delta G 0 prime that is effectively 0. And so cells basically match their ATP to ADP ratios with any other ratios of nucleoside triphosphates and diphosphates because of this reaction. And so that's why the two GTP are roughly an ATP equivalent. So we'll just call it 6 ATP plus 2 NADH to reverse the GAPDH step. That gives us 6 ADP plus 2 NAD+ plus 6 inorganic phosphate plus a glucose. The delta G 0 prime for this entire coupled pathway is on the order of negative 9 kcals per mol, with the delta G less than 0, equilibrium lies to the right. Net synthesis of glucose is favorable. And so if we just think about this in ATP terms, remember, when we discussed glycolysis, you harvest net two ATP from conversion of glucose into two pyruvate. If we reverse the reaction doing gluconeogenesis, in ATP terms, we have to invest six ATP to turn that pyruvate back into glucose. Makes sense. We didn't make a perpetual motion machine. Both pathways are favorable. But it costs more energy to make a glucose than we can get out by burning the glucose. I should say, the energy isn't just the ATP. There's also this NADH.

~16:23That NADH that's produced from glycolysis also is storing energy, if you will, from oxidation and an NADH/NAD+ ratio, and you're consuming that going in the opposite direction. You only do gluconeogenesis if you have a high ATP:ADP ratio, of course, and a NADH/NAH+ plus ratio-- that is, if you have it under the right conditions to actually run this pathway and have it make sense for the cell. All right. Now I want to discuss, briefly, how these enzymes in gluconeogenesis work. I'm not going to spend time on fructose 1,6-bisphosphatase or glucose 6-phosphatase. As I said before, this is just hydrolysis of that phosphate-alcohol bond. That's relatively straightforward. But I do want to spend time on pyruvate carboxylase and PEP carboxykinase because this introduces a new reaction, carboxylation. But it also introduces a new concept in metabolism that is actually quite important in eukaryotic cells. And that is this issue of so-called compartmentalized metabolism. And, of course, you've learned in introductory biology that a big difference between eukaryotes and prokaryotes is eukaryotes have all of these organelles-- that is, these membrane-bound structures within cells that carry out various functions. So one reason that it's useful to have these different membrane-bound organelles is it creates different compartments within the cell. What does that mean? Well, if you have different compartments, that means you can create different conditions within each compartment. And that's very important for metabolism. Well, why is that important for metabolism? Well, because, remember, whether or not reactions are favorable depend on delta G, which, of course, is dependent on the equilibrium constant, which is related to delta G 0 prime but also to the ratio of the products and reactants within that compartment. And so by having different compartments, you can have different ratios of metabolites-- say, a different ratio of ATP to ATP or a different ratio of NADH to NAD+. Having those different ratios means that because delta G is proportional to the ratio of the reactants over the products-- I'm sorry-- products over reactants, by having different ratios of things like ATP and ADP, NAD, NADH, you can make different reactions more or less favorable depending on which compartment in the cell that they're located. Now it turns out that glycolysis and most of gluconeogenesis takes place in the cytosol of eukaryotic cells. The cytosol is basically the space inside the cell that's not inside another organelle. However, pyruvate carboxylase is a reaction that takes place in mitochondria because mitochondria have a particularly high ATP:ADP ratio. And it turns out, this helps favor the pyruvate carboxylase reaction. And so we'll talk a lot about reactions in the site cytosol versus the mitochondria. But basically, here's glucose to PEP. Whether you do that in one direction by glycolysis or the other direction by gluconeogenesis, that happens in the cytosol as does turning that PEP into pyruvate. But if you're going to reverse the reaction, eukaryotes take advantage of a different compartment, the mitochondria, with a high ATP to ADP ratio. And basically, in the mitochondria, turn that pyruvate into oxaloacetic acid-- that is, carry out the pyruvate carboxylase reaction. And then that pyruvate carboxylase reaction generates oxaloacetic acid, and then PEPCK can, either in the mitochondria or in the cytosol, generate PEP. And so here's a place where one can run one reaction-- PEP to pyruvate of t make ATP in the cytosol-- but a different reaction-- pyruvate carboxylase to turn pyruvate into oxaloacetic acid that would then be used for PEPCK to turn that back into PEP and have that take place in the mitochondria.

~19:39I want to point out, just so for MCAT exams and things like that, PEPCK is classically defined as a cytosolic activity, at least in terms of gluconeogenesis. Although, there is a mitochondrial isoform of PEPCK. And it's at least somewhat debated. There's some evidence that, at least, in some tissues, that might be important for gluconeogenesis as well. And so that's why I draw it as PEPCK using oxaloacetate to PEP either in the mitochondria or in the cytosol because that's an area of active investigation right now. All right. Let's start by talking about how does pyruvate carboxylase work? This is an example of a carboxylase reaction, that is, adding a CO2 group to a molecule to make a carbon-carbon bond. That purple CO2 is added to the CH3 of pyruvate of to make oxaloacetate. And how this happens, in many cases, uses a cofactor. And that cofactor is called biotin. Biotin, like many cofactors, is a vitamin. And this provides a useful functional group. In this case, enzymes that use biotin use ATP to basically drive CO2 addition to biotin. And then that CO2 on the biotin is then activated to be transferred to another molecule in a carboxylation reaction. All right. So what does biotin look like? I will draw it for you. So this, here, would be biotin. I've drawn it in the enolate form of biotin. I'll draw it in a different way in a second. Biotin is typically bound to a lysine molecule in the active site of an enzyme that uses this. And so this, here, is basically a peptide bond between the terminal amino group, the epsilon amino group of the side chain of a lysine in a residue that basically makes a peptide bond to link biotin into the active site of the enzyme that's using it. Biotin is often drawn in the keto form. And the active part of biotin is basically this top part of the molecule. And so to draw it to see differences between the enolate and keto form, I just want to show that quickly. I drew it in the enolate form because it's easier to see, I think, how it-- so this, here, would be the keto form where biotin is drawn most commonly. And basically, the reaction, the way biotin picks up a CO2 is as follows. And that is CO2 can exist particularly under basic conditions as CO2 or as-- this is bicarbonate. OK. So bicarbonate-- turns out, some enzymes use CO2 directly. Some enzymes use bicarbonate. It turns out that pyruvate carboxylase uses bicarbonate. So biotin bound in the active site of pyruvate carboxylase will utilize bicarbonate in the following way. And I'll just point out that the pH of the mitochondria is also more basic, which also helps this pyruvate carboxylation reaction. All right. So what happens is this bicarbonate is phosphorylated by ATP. This phospho intermediate can then react with this active site here of biotin to release the phosphate. And now, you now have this activated CO2 group that's attached to the biotin. Here's pyruvate drawn in the enol form. You can look back in your notes about how to interconvert it between keto and the enol form. I've shown that several times. And then this, then, can end up adding this CO2 to the end of pyruvate to generate oxaloacetic acid plus regenerate the biotin cofactor in the right form. It'll be the keto form, which can then get back to the enol form. And it's ready to do another catalytic cycle. And so, basically, pyruvate carboxylase uses ATP to produce this carboxylate of biotin. And then uses that to add the CO2 to pyruvate to generate oxaloacetic acid. Now I want to point out that oxaloacetic acid is both an alpha keto acid like pyruvate was. And it's a beta keto acid. And so this ketone is alpha to that carboxylic acid. And it's beta to that carboxylic acid. So it's both an alpha and a beta keto acid. And it turns out that the way PEPCK works is it takes advantage of the favorable decarbonization of a beta keto acid, which we talked about last time, along with GTP to phosphorylate pyruvate and trap it in the enol form.

~23:33And so this is how PEPCK works. So I'm going to draw GTP in this very stylized way-- so guanine with three phosphate groups. There's oxaloacetate. That decarboxylates-- takes advantage of this being a beta keto acid to decarboxylate and add the phosphate. And that's how we can generate PEP. OK. Great. And so that's how you can build a different pathway that uses ATP and GTP for energy input in order to run glycolysis in the opposite direction-- gluconeogenesis. All right. So how is gluconeogenesis regulated? Well, just like the principles we talked about in glycolysis, it works in a way that makes sense. And so you can guess that the steps that are going to be regulated are exactly the ones that you might guess. They were the same steps that were regulated running glycolysis. It's going to be these big changes where energy changes occur across the pathway, which are also the entrance and exit of the pathway. And basically, regulation has to be reciprocal. So in glycolysis, you want to increase glycolysis under conditions where you need to produce ATP. And you want to decrease glycolysis if you have enough ATP or enough of another downstream product, such as citrate. All right. Well, gluconeogenesis is going to be exactly the opposite. You certainly don't want to do gluconeogenesis if you need energy release. If you need ATP, you don't want to run gluconeogenesis. However, you want to increase gluconeogenesis, if the cell has ATP excess, you also want to do it if you have excess of other products like citrate because why go through the trouble of sending things down glycolysis if you have nowhere to put it. You might as well, instead, make glucose or shunt that glucose off to produce glycogen. And so let me just add that regulation here. And so major regulators of gluconeogenesis are as follows. And so high levels of citrate is going to stimulate fructose 1,6-bisphosphatase. So high levels of citrate will inhibit things coming through glycolysis and activate gluconeogenesis by acting at fructose 1,6-bisphosphatase to match the energy considerations. If you have high levels of AMP, energy charge is low. You want to stimulate glycolysis at phosphofructokinase. Similarly, that's going to inhibit gluconeogenesis at FBPase. And it turns out, high levels of ADP-- I'm sorry-- yeah, high levels of ADP, low energy charge, also inhibits PEPCK because you also don't want to try to generate PEP under those conditions. Great. All right. Now in animals, we also want to regulate glucose catabolism and production under signaling control. And this gets back to this other topic we talked about, such as the liver's job being to maintain a constant level of blood sugars. Most of you are very familiar with blood sugar control through diabetes. You've probably heard that this is under hormonal control, that the job of insulin is to lower blood glucose. And you also have hormones like epinephrine or glucagon whose job is to raise blood glucose. Why do you want to raise blood glucose? Well, if you have some kind of fight-or-flight response-- you see that lion, and you need to run away, your body has this adrenaline rush. That's what epinephrine is. That basically wants to supply more energy to your muscles, so that you can run away effectively. So it turns out, both insulin and glucose act on lots of tissues in the body. I'm going to focus on what they do in the liver. And that's because in the liver, this is a major organ that regulates blood glucose levels. So if you have excess glucose around, what do you want to do? You want to stimulate glucose uptake into cells, its metabolism, and its storage. And so insulin with high blood sugar basically wants to stimulate, taking glucose into the liver and storing it as glycogen.

~27:03If you want to run away from the lion, well, now you have epinephrine around. You want to make sure that as you're consuming that glucose in your blood that you're continually making more. And so you want the epinephrine to stimulate release of glucose either from gluconeogenesis or from the breakdown of glycogen. And both of these hormones act-- insulin and epinephrine-- act at a level where they can regulate enzymes of glycolysis and gluconeogenesis as well as entry and exit of glucose monomers in and out of glycogen. And so this is an example of how fructose 2,6-bisphosphate works. And so remember you learned from Professor Yaffe that epinephrine acts via cyclic AMP signaling, which turns on a kinase-- protein kinase A. And that protein kinase A can regulate enzymes that produce or break down, basically, these enzymes that produce or break down fructose 2,6-bisphosphatase which, in turn, regulates PFK activities such that you can match your need to burn that glucose versus doing gluconeogenesis in the case of the liver, so you can do enough gluconeogenesis in order to have glucose around for the body to use in that fight-or-flight response being driven by epinephrine. Now a major effective insulin and epinephrine signaling, though, is actually on release or storage of glucose molecules in glycogen. And I alluded last time in talking about the regulation hexokinase that glucose 6phosphate is basically the entry point to get glucose units in and out of glycogen. And I want to discuss how you add and subtract those glucose units into storage polymers. Now I'm going to discuss it in the context of glycogen storage in humans. But remember, plants actually store things also as glucose polymers. The chemistry is very similar. But, obviously, the regulation is very different.

~28:41Now you'll hopefully recall from past lectures that these storage polymers of glucose-- glycogen in humans, starch in plants-- are basically these alpha 1,4 linkages of glucose molecules. Remember, there was a nonreducing end and a reducing end of the molecule. So starch was a straight-chain polymer. Glycogen had these alpha 1,6 branch points that made this branch polymer, where there's lots of non-reducing ends and a single reducing end. And each of these non-reducing ends is a polymer of glucose molecules with this alpha 1,4 linkage that, at the branch point, has this alpha 1,6 linkage to make this long-chain branched polymer. Now when we discussed this at the time, I mentioned that this is useful because you have all these non-reducing ends that can be used to add or subtract glucose monomers. And that's great. This is a nice compact form of storage with lots of places to put or remove glucose from. You either store it quickly, insulin driving glucose storage-- or remove it quickly, epinephrine driving release of glucose from the glycogen. And so if we're going to have a way to add glucose polymers or break them down, those are two separate pathways, just like glycolysis and gluconeogenesis-- reciprocal activities, but we need two pathways to do it because delta G has to be less than 0 for each pathway to work. Also, we have to be able to regulate these separately because we don't want to have a futile cycle. Now the way you add and subtract glucose polymers to glycogen acts through glucose 6-phosphate being first converted to glucose 1-phosphate. So this, here, is alpha glucose 6-phosphate. Remember, it's alpha because I drew that hydroxyl at the one position pointing down. This, obviously, can enter glycolysis and come in from glucose via hexokinase. All right.

~30:19This is first converted via a mutase reaction to move the phosphate from the 6 to the 1 position of glucose. So this, here, is glucose 1-phosphate. The way the mutase reaction works is exactly the same analogous mechanism that I explained for phosphoglycerate mutase in glycolysis-- moves the phosphate in this case from the 6 to the 1 position of glucose. And then once you have that glucose 1-phosphate, that can be added to a non-reducing end of a glycogen polymer using an enzyme called Glycogen Synthase, GS. So that'll give me a glycogen molecule with one additional monomer added to the non-reducing end of the polymer. And you release that glucose back off via a different pathway, a different enzyme called phosphorylase. All right. Now the activity of these enzymes-- glycogen synthase and phosphorylase-- that is, the pathway to add glucose 1-phosphate glucose monomer to the polymer versus remove it from the polymer is under hormonal signaling control in animals. And basically, that hormonal signaling works as follows. So phosphorylase can be phosphorylated or dephosphorylated via a signaling enzyme, an enzyme like phosphorylase kinase, which is downstream of PKA, which is downstream of epinephrine signaling. OK. And so when phosphorylase is phosphorylated by phosphorylase kinase-- that has, has a phosphate group added is a signaling cascade-- it is in the active state. And when it's dephosphorylated, it's in the inactive state. Well, glycogen synthase also is subject to regulation by protein phosphorylation on the enzyme by a kinase. Except this has the opposite relationship. So when it is in the phosphorylated state, it is inactive. But it is in the non-phosphorylated state, it is active. And so protein kinase A, which is downstream of epinephrine, can both activate phosphorylation of phosphorylase and glycogen synthase. And that makes sense. You want to release glycogen glucose monomers from glycogen. You turn on, by phosphorylation, the pathway to release them. And you turn off the pathway to store them. Another kinase that phosphorylates to glycogen synthase is a kinase called GSK or Glycogen Synthase Kinase. This kinase is inhibited by insulin signaling. And so this is a negative of a negative. So effectively, a negative of a negative will keep it in the inactive state. And so insulin, by inhibiting the ability of GSK to put glycogen synthase in the inactive state, will make glycogen synthase active. And when you have high insulin around, that actives glycogen synthesis, and you store glucose monomers as glycogen. Get them out of the blood. OK. Now let's discuss the chemistry that allows us to do this glycogen synthase and phosphorylates reactions. Now as we go through this, what you will see is that when we break down the polymer, energy is released. So that's going to be breaking down a polymer. That's the right direction of entropy. And so that's going to be released. And storing it is going to require energy. That is, you're going to have to have some energy input to build a polymer. But nature actually does this in a way that is actually quite efficient. First, let's talk about how you store glucose 1-phosphate units by adding them to glycogen. So this uses energy input from UTP, which is an ATP equivalent for exactly the reason I described earlier. That is, because ATP plus UDP, like any other interconversion of nucleoside triphosphates and diphosphates is very close to equilibrium.

~33:26So the UTP to UDP ratio should be similar to the ATP the ADP ratio. And so UTP is really an ATP equivalent. But nature, for whatever reason, decided to use UTP here. And it generates a nucleoside sugar called UDP glucose. And so let me show you what that is. So here is glucose 1-phosphate. I will draw a stylized version of UTP here. And so this molecule with the UDP added to the glucose is UDP glucose. This will also generate a pyrophosphate. And that pyrophosphate can be hydrolyzed to generate two inorganic phosphates, effectively, like building any other polymer, by doing this downstream-- keeping that product of the first reaction low helps pull that reaction forward for polymer synthesis. This UDP glucose can now react with the non-reducing end. So this, here, will be the non-reducing end of the polymer. That's the OH group at the 4 position, at the nonproducing end. And so that then generates that alpha 1,4 bond to add to the polymer. So here, we have glycogen n plus 1 plus releases UDP. And so the net to add a glucose 1-phosphate to the non-reducing end of the growing glycogen polymer is net conversion of a UTP to a UDP. So that is one ATP equivalent to add glucose 1-phosphate to the end of the polymer. Now, of course, you had it to also have an ATP from hexokinase to capture that glucose 6-phosphate to begin with. But effectively, this is two ADP molecules to add a glucose to glycogen. All right. Now if we're generating starch, that's all we have to do, just build a long polymer. But if we're making glycogen, remember, glycogen has all of these branch points on them as well. And to do the branch points, you basically have to make this alpha 1,6 bond, which then gives you two new non-reducing ends that you can add more subunits to. The way nature does this is as follows. And so if I just draw this here in a stylized way, it turns out, once you get to about seven units here in the growing polymer-- so this is the reducing end, and this is the non-reducing end of the polymer. And so the polymer is growing by adding things to that end. Once you get about seven, there's an enzyme that will basically cleave this, and move it and make a new alpha 1,6 bond. OK. Here we go. We have our reducing and our non-reducing end and basically transfers these 7 units over. And now, we have two non-reducing ends that we can continue to grow the polymer from. You can think of this like the airplane analogy. When you load an airplane, at least the way that's most efficient, the people who are sitting in the back of the plane, they get on first. But then, they're the last people off the plane because you add basically to these non-reducing ends, and then you subtract from those non-reducing ends. And so the first glucose added, the one at the reducing end, is the last one that is going to be removed. All right. Now phosphorylase is the enzyme that allows you to do the opposite-- that is, to break that alpha 1,4 bond and release monomers. And it's called phosphorylase because it uses phosphate to break that alpha 1,4 bond, taking advantage of the fact that breaking down the polymer is favorable and actually releasing the glucose from it again is a glucose 1-phosphate. And so at the non-reducing end of the molecule-- so this, here, would be the non-reducing end of the glycogen polymer. You basically have a phosphate molecule that takes that off, so you have a glycogen n minus 1 plus a glucose 1 phosphate, which can now undergo a mutase reaction back to glucose 6-phosphate that could then be released from the cell if it's a liver, or it could be burned in glycolysis.

~37:02Now, obviously, if you're doing breakdown, that is, your chewing back from non-reducing ends, eventually, you're going to hit a situation where you come to where there's just a nub here with an alpha 1,6 bond there. That is, chew it back till you hit this single monomer with a non-reducing end here to this alpha 1,6 break point. Turns out, you just cleave off that nub, if you will. And so that nub is then just released as free glucose. So other than release of that nub, what it means is that you actually basically get no ATP to actually break down in the phosphorylase reaction. So the net cost to store a glucose 1-phosphate molecule is one ATP via the UTP to UDP conversion in the glycogen synthase reaction. And you get that one glucose 1-phosphate back. And so two ATPs to put a glucose tin glycogen. And then you get it directly back out as a glucose 1-phosphate. That's a phosphorylated glucose that can then enter glycolysis. So that's one less phosphate you need to spend on glycolysis. And so it's incredibly efficient to store glucose in that way. And so it's actually slightly greater than one ATP to store it as glycogen because you lose in ATP with that nub removal at the branch polymer. But for all intents and purposes, it costs one ATP to store your glucose as glycogen, which is pretty amazing. All right. Now before I leave the topic of sugar storage, I want to make some comments on some sugars that are stored in forms other than glucose. And want to talk about these because these sugars are, of course, important parts of our diet. And so as we've mentioned in prior lectures, two of these sugars are sucrose and lactose. So remember, sucrose is a disaccharide of glucose plus fructose. And lactose is a disaccharide of glucose plus galactose. So those are storage forms of sugar-- sucrose in the case of plants, lactose in the case of milk made by mammals. And so to break those sugars down, basically, you break the disaccharide. Now, you have the glucose molecules you know how to handle. That's glycolysis. But I want to spend a little bit of time how you deal with the fructose and the galactose that you get out from these sugars because this actually relates to some things that I'm sure you've all read about in the news. For instance, fructose as a sugar in our diet is actually quite controversial. Lots of stuff out there that says fructose was cooked up in the devil's kitchen and is this evil thing. There's actually others who argue that, oh, fructose is the same as glucose, the same number of calories. There's both sides of the debate. Both sides are actually making factual claims. And I want you to understand the biochemistry, so you can actually judge for yourself who's right in those claims. All right. So let's start with talking about how you metabolize fructose. And so fructose is first captured by phosphorylation to generate fructose 1-phosphate. This is carried out by an enzyme called ketohexokinase. Ketohexokinase is an enzyme this present in your gut and your liver. And so fructose in the diet, from breaking down sucrose or from high-fructose corn syrup, whatever, is captured with ketohexokinase to make fructose 1-phosphate. Turns out that phosphofructokinase, our old friend from glycolysis, is less efficient than ketohexokinase but can also carry out this reaction. And remember, what is it do in glycolysis? It adds a phosphate to the 1 position of fructose 6phosphate. Well, it can also add a phosphate to the 1 position of fructose give you fructose 1-phosphate.

~40:24OK. Once you have this fructose 1-phosphate, which, remember, is not in glycolysis, it can be a substrate for the glycolytic enzyme aldolase. What does aldolase do in glycolysis? It splits FBP into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Well, here, you only have a phosphate on the 1 position. And so if you look back at how aldolase works, what you'll see is that if aldolase acts on fructose 1-phosphate, you'll generate a dihydroxyacetone phosphate. That phosphate will go to the dihydroxyacetone half, which, of course, is perfectly good substrate for glycolysis. The other product of aldolase acting on fructose 1-phosphate is just glyceraldehyde without the phosphate on it. And then there's another enzyme that will phosphorylate glyceraldehyde to generate glyceraldehyde 3phosphate, which can then also, of course, enter glycolysis. And so fructose is a hexose just like glucose. You generate two trioses-- DHAP and glyceraldehyde 3-phosphate. That costs you two ATP to do it, exactly the same cost is what you spent to get glucose to make DHAP and glyceraldehyde 3-phosphate. And so people are absolutely right when they say metabolizing fructose has exactly the same caloric cost as metabolizing glucose-- two ATPs to get it into the trioses. And then whatever you get out of it, you get out of it two ATPs, four ATPs-- so net two ATPs if you just do fermentation, more if you do complete oxidation. But the bottom line is that from that standpoint, you get exactly the same number of calories from burning glucose versus fructose. However, I want to point out that the regulation will not be the same. And that's because in this fructose metabolism, you will notice that there was no glucose 6-phosphate and no FBP generated. That means you are avoiding all of the feedbacks that control how much glucose you send to glycolysis-- first to glycogen. Remember, that's what glucose 6-phosphate does, acting on trapping sugar. And more importantly, you're actually not using phosphofructokinase in a way to fit with the rest of the feedback regulation. I also show that here on this slide. It's basically a better version of what I drew up here. And I now want to point out this storage of citrate and this feedback regulation of citrate. So we'll talk about the specifics of this more in other lectures. But it turns out, citrate is the precursor for how one stores carbon as fat. And so if you're putting fructose glucose into the system and citrate is too high, it'll shut off phosphofructokinase and stop carbon coming down the system, shunting it instead to glycogen or just saying don't give me any more glucose at all. If I'm doing the same thing with fructose, those feedbacks don't exist. Citrate doesn't feed back on any of the enzymes that are regulated for fructose metabolism. And so the idea is carbon keeps getting dumped into citrate, which ultimately ends up as fat. And this really underlies, at least on a theoretical basis, why there may be a connection between fructose and obesity or metabolic syndrome. I've posted articles on Stellar, one from The Popular Press, one from a scientific journal that discusses the controversy, discusses this. But now, at least, you have the background to draw some of your own conclusions. OK. That's fructose metabolism. What about galactose metabolism? Well, galactose metabolism interfaces with, effectively, glycogen metabolism or, at least, glucose 1-phosphate-or sorry, UDP glucose and does so in a way that is a little bit non-intuitive. But it's actually very important to understand galactose metabolism, at least for those of you who are looking to go to medical school.

~43:42It turns out that galactose metabolism and issues with metabolizing galactose are what underlie a set of rare genetic diseases leading to a set of conditions called galactosemias. I guess, if you're a pediatrician, you may encounter these. If you're not, these are quite rare. Although med schools love to ask about these, and certainly, they're very popular on things like board exams. So it's something you should know about. And so I just want to briefly introduce galactose metabolism to you.

~44:09So this is galactose. Like all sugars, galactose is initially trapped by phosphorylation. It's phosphorylated on the 1 position to trap it, just like fructose. And so this is galactose 1-phosphate. Remember, galactose is an epimer of glucose. So it differs from glucose here by the stereochemistry of the hydroxyl group at this 4 carbon. And so the way galactose, once it's trapped as galactose 1-phosphate is metabolized is it will react with UDP glucose. So here is UDP glucose from glycogen metabolism drawn in a very stylized way. That will generate a UDP galactose and a glucose 1-phosphate, which, of course, can enter glycolysis, can be stored as glycogen, et cetera. This UDP galactose can then be turned into UDP glucose via an epimerase reaction. And so remember, the epimerase reaction needs to change the stereochemistry of the hydroxyl group at this 4 position, such that it's pointing up in galactose or pointing down in glucose. The way epimerases work is that they require a cofactor. That cofactor is NAD. and they work in the following way. And so if I just draw, here's the 4 position of galactose, the 4 carbon of galactose, with the hydroxyl group pointing up, an epimerase can basically remove a hydride from the bottom face of the molecule, transfer those two electrons to NAD to generate NADH. Now, you get this lactone intermediate at the 4 carbon. And then you can have two electrons from an NADH can be added to the opposite face of the molecule that regenerates an NAD+. And now, we've effectively changed the stereochemistry at the 4 carbon to now be pointing such the hydroxyl points down, and that's glucose. And so the way epimerases work is they basically remove and add electrons from different faces of the pyranose ring. And that changes the stereochemistry of which direction the hydroxyl group is pointing. And so effectively, this is how we metabolize all kinds of different sugars, either putting them in glycogen or other starch, some other storage carbohydrate that we can get them later, and then use them in glycolysis as a way to drive glucose oxidation as a way to release energy and keep ATP high in the cell. All right. Now I want to shift topics and begin to discuss how we can oxidize pyruvate from glycolysis to make CO2 as an alternative to how we can release energy from carbohydrate oxidation rather than just use fermentation. Now you'll see that the enzymes and ways that we do this is going to also be how we make citrate. And so this isn't just about releasing energy. It's also about storing energy because you'll see, citrate is a precursor for storage as fat. The reactions downstream of pyruvate oxidation also make lots of other useful intermediates that can be used to synthesize other things cells need-- amino acids, nucleic acids. And so this pathways we're going to discuss are going to come up over and over again throughout the course. Now to facilitate this discussion, I'm really going to at least initially focus on catabolism.

~46:58And so just to remind you, if we do glycolysis by turning glucose pyruvate, this involves oxidation. So we need to dispose of those electrons somewhere. In other words, that NADH has to be regenerated NAD. Fermentation of pyruvate into something like lactate is a way to do that and allows net ATP production from glycolysis without the need for any oxygen. However, if we're going to completely oxidize that pyruvate CO2 we're going to generate many more electrons as waste that we have to put somewhere. Oxygen is a particularly good electron acceptor. And so we can reduce oxygen to water as a way to deal with that electron waste, and that allows us to, just burning wood, release lots of energy from glucose by transferring those electrons ultimately to oxygen. And of course, that same process-oxygen to water-- if we're using the pyruvate to fully oxidize it, also is necessary to regenerate the NAD to run glycolysis. Now ultimately, the series of reactions that allow chemically conversion of pyruvate to CO2 is called the TCA cycle for try Tricarboxylic Acid Cycle or sometimes referred to as the Krebs cycle and also is referred to as the citric acid cycle-- so TCA, Tricarboxylic Acid Cycle, Krebs cycle, citric acid cycle-- all synonyms for the same thing. What these cycles are is basically a way to take two carbon units. And those two carbon units can come from the oxidation of pyruvate or from the oxidation of lots of other fuels. And those two carbon units are combined with four carbon units. You'll see oxaloacetate is the four-carbon unit-- oxaloacetate gluconeogenesis. That oxaloacetate could come from the pyruvate-carboxylase reaction. Or it could come, as you'll see, from the TCA cycle itself. This generates a six-carbon molecule, which is citrate. Citrate is a tricarboxylic acid, hence the citric acid cycle or the tricarboxylic acid cycle. And then those six carbon units are reoxidized, releasing two CO2 molecules, back to four-carbon oxaloacetate. And hence, this is a cycle. Now this cycle was initially described by Hans Krebs, who predicted the cycle largely based on chemistry-- one of the most amazing feats in the discovery of metabolism. Turned out, he was right. And thus, it's also referred to as his name, the Krebs cycle. Now lots of oxidation is happening in this cycle. You're oxidizing the molecule, releasing CO2. That means lots of energy is released. Those electrons ultimately will be transferred oxygen. And so we can use this to make lots of ATP and do lots of other work. Where are these reactions all occur is in the mitochondria, specifically in the matrix of the mitochondria. Remember, the mitochondria, from cell biology, is a double-membrane organelle. The innermost part of the mitochondria is referred to as the matrix. And these have conditions that favor pyruvate carboxylase reaction, as I talked about, but also favor other oxidation reactions, such as those that occur in the TCA cycle. And so next time, what I will start with is by being more explicit about what chemistry is happening to allow these two carbon units to combine with oxaloacetate 4-carbon to generate citrate and go over the reactions that then allow you to run a cycle to regenerate oxaloacetate, that is, run the TCA cycle as a way to oxidize carbon. Thanks.

🎬 MIT 7.05 Lec 23 · TCA Cycle II, Isotope Tracing & Glyoxylate Cycle⏱ ~50:00

~00:00MITOCW | 23. TCA Cycle II

~00:02MATTHEW

~00:02Hello, everybody. Last time, I introduced the idea of the TCA cycle, tricarboxylic acid cycle. Also known as the

~00:10VANDER

~00:10citric acid cycle, because citric acid is a tricarboxylic acid, as you'll see later today. Also known as the Krebs

~00:18HEIDEN:

~00:18cycle, named after Hans Krebs, who discovered it in the early part of the last century. The TCA cycle is the series of reactions that occurs in the mitochondrial matrix and it allows the complete oxidation of two carbon units, derived from many things, including pyruvate, derived from glucose and glycolysis and enables the complete oxidation of that carbon to CO2. Now, it's a cycle because those two carbon units from pyruvate or other sources enter the cycle, combine with 4carbon oxaloacetate, and combine to make 6-carbon citrate. Hence the citric acid cycle, or TCA cycle, that is then oxidized back to 4 carbon units, forming a cycle that allows cells to release lots of energy. Complete oxygen enables, ultimately, complete oxidation of glucose to CO2. We've talked many times how this releases energy. And it also generates lots of intermediates for the cell that can be used to make other stuff. Last time, I alluded to the fact that citrate can be used, say, to make fatty acids. Today, what I want to do is I want to dive into the details of how this TCA cycle occurs and the consequences of the way it works, and how that affects other aspects of metabolism. You'll see that it actually affects the ability of different organisms, whether or not they can make things from intermediates in this cycle, because of how the cycle works. Now, to start. Of course, if we're going to start from pyruvate. Pyruvate, of course, has three carbons. And so if we're going to generate a two-carbon acetate group, we have to lose CO2. And as I described a couple lectures ago, acetate is also the thing that we can derive from metabolism of ethanol, showing us two things-- metabolized glucose to pyruvate or ethanol itself-- can both be turned into acetate. But really, the donor is this molecule, acetyl-CoA. Which is basically also a carboxylic acid, but rather than being a carboxylic acid, instead makes this a thioester bond, which activates this carbon as a leaving group, such that it can combine with oxaloacetate, which you'll remember from gluconeogenesis, releasing that S-CoA molecule to generate six-carbon citric. So, this is citric. You can see that we've made a bond from this carbon on the acetate, losing this S-CoA group to this carbon oxaloacetate to make this six-carbon citrate molecule, which is a one, two, three-- three-carboxylic acid, or tricarboxylic acid. Hence the name citric acid cycle, tricarboxylic acid cycle.

~02:58Now, these six carbons can then-- or this six-carbon citrate molecule can then be oxidized, generating two CO2 molecules that are released, and ultimately reforming oxaloacetate that can pick up another two-carbon acetylCoA to generate another citrate, and around and around the cycle goes, allowing in the end the net entry of two carbons, effectively from acetate, and release of two carbons as CO2. Now, as I alluded to, this can come from pyruvate. It can come from acetate itself, vinegar. It can come from alcohol. It turns out that when you break down fat, you also break it into two carbon units. And so this cycle becomes very useful for cells, because it allows the oxidation of many different molecules to completely turn that carbon into CO2. Now, if we're going to do this from glucose, however, you'll remember that pyruvate has three carbons. And so if we're going to turn pyruvate into acetate, or acetyl-CoA, we have to lose a carbon of CO2. We have to lose this carbon as CO2. Now, we saw this before, that we can do this via-- this is exactly how we generated ethanol when we did fermentation of pyruvate to ethanol, but that molecule didn't generate acetate, It generated acetaldehyde. The difference, of course, being whether or not this carbon gets oxidized in the case of acetate to the acid, or in acetaldehyde in ethanol metabolism, where it remains an aldehyde. Now obviously, you can make ethanol and then turn that ethanol into acetate. And that's a pathway that I guess certainly would work. However, that's not the way it happens in most organisms. Most organisms actually directly produce acetate, or more correctly, acetyl-CoA from pyruvate. So let's take a look at that reaction. So here once again is pyruvate. And if we turn that pyruvate into this two-carbon acetyl-CoA, let's look here what has to happen. Well, the first thing is that we have to decarboxylate this molecule. And so that generates a CO2. We have to lose that one carbon. Also, as I pointed out, if we do this as if we did it in ethanol metabolism, we'd be left with an aldehyde. But this is an acid, and so this carbon also has to be oxidized. We know how to do that. We can donate those electrons to something like NAD, so the NAD is reduced to NADH. And we had to add this S-CoA molecule, which I'll come to in a minute. And so each of these steps ends up making a fairly complicated reaction. Obviously, several co-factors are going to be involved. You should be able to guess that just by looking at it. Obviously, I already drew up there NAD. If we're going to decarboxylate, remember this is a alpha carboxylic, an alpha ketoacid-- the ketone group is alpha to the carboxylic acid. And so if we're doing alpha decarboxylation, as you might guess, we need a cofactor. That co-factor, as I told you before, is the co-factor factor thiamine pyrophosphate. And finally, there's this S-CoA group. We need to describe what that is. However, before delving into those, and there's actually a couple other factors that are needed as well, I want to mention one other issue about this reaction, in that this reaction happens in the mitochondria, because that's also where the TCA cycle happens. So, recall that if we divide the cell into two compartments like an eukaryotic cell, here we have the cytosol and the mitochondria in a eukaryotic cell. As we've already talked about, having different compartments helps facilitate different metabolic reactions, because you can have different conditions in the two compartments.

~06:54And so as we said, glycolysis occurs in the cytosol. Glucose to pyruvate. And last time I mentioned, the TCA cycle occurs in the mitochondria. And so that means if we're going to fully oxidize the pyruvate carbon to CO2 using the TCA cycle in the mitochondria, that pyruvate has to get from the cytosol to the mitochondria, or at least carbons from the pyruvate have to get there. It turns out, you'll see in a minute, acetyl-CoA is a very large group. And so pyruvate itself is transported into the mitochondria. And that's where the reaction occurs to turn it into acetyl-CoA that can then enter the TCA cycle and oxidize. However, what this means is that a transporter is actually needed to get this across the mitochondrial membranes and into the matrix of the mitochondria where the TCA cycle happens. And I like to mention this because it turns out the pyruvate carrier-- that is, the transporter-- the way that it actually gets that pyruvate from the cytosol into the mitochondria actually was an unknown thing about metabolism until 2012, so not that long ago. Sometimes one can be left with the feeling, listening to these metabolism classes or reading a textbook, that everything about metabolism has been known forever, but it's actually not true. Here's a very key, central part of the pathway that was actually just discovered, at least at the time of this lecture, less than 10 years ago. It also illustrates that not all metabolism is completely understood. Now, let's get back to this reaction, how you turn pyruvate into acetyl-CoA. And I want to go through now and point out that several co-factors are needed. And so I already mentioned one of them, S-CoA, which is shorthand for coenzyme A. So, I need to tell you what that is. You hopefully are already familiar with TPP plus, thiamine pyrophosphate. We talked about that when we talked about how you do alpha decarboxylation to generate ethanol from pyruvate, also used here for the alpha decarboxylation reaction. Redox reaction happens, and so we needed NAD plus to get converted into NADH. We talked a lot about how that serves as an electron carrier, but it turns out that there's two additional electron carriers that are involved in this reaction. One of them is called FAD and the other one is called lipoic acid. Now you might say, why do we need all these electron carriers? Well, these are just different molecules that can carry two electrons, similar to NADH. And effectively, what these can do by having multiple electron carriers-- one can build chains of oxidation and reduction reactions. And it turns out these chains of oxidation reduction reactions really become central to energy transfers in biology, because building these chains allows more easy stepwise release of energy as one moves across these oxidation reduction reactions. Which remember, as I alluded to earlier, really are at the core of bioenergetics and a lot of what allows energy release from these pathways. What I mean by this will be more explicit as we go through what some of these co-factors look like. So, I'm not going to draw TPP plus or NAD again, but let's define what some of these other cofactors look like. So, let's start with coenzyme A. So, coenzyme A, it turns out, is useful. It's actually involved in lots of acylation reactions.

~10:34What's an acylation reaction? Well, that's basically if you're making a carbon-carbon bond by adding a molecule of greater than one carbon, so two carbons or greater, to something else. That's an acylation reaction, as we did with adding the two-carbon acetate to oxaloacetate to make citrate. And you'll actually see coenzyme A will come up in this in many, many lectures throughout the rest of the course.

~11:00Why this becomes useful is because it activates this basically carbon to the left of, in this case, the ketone. Because, by having that thioester there, ends up activating that carbon and allows it to carry out these acylation reactions. Coenzyme A itself is derived from a vitamin called pantothenic acid. And as a vitamin, this is something that you have to get from the diet. Now, when we abbreviate coenzyme A, which is often abbreviated S-CoA, you get the sense that it's this little tiny molecule that's just stuck on sulfur-- stuck on to the end to make this thioester bond. But it turns out coenzyme A is actually a giant molecule, one of the reasons why you actually synthesize acetyl-CoA in the mitochondria, because it's hard to transport this giant molecule. And so this is what pantothenic acid looks like. So, if I put an acid group here and a hydroxyl group there, that would be pantothenic acid, the thing that's in your cereal, on the side of your cereal box. Then there's these additional pieces. This is the active end of the molecule, that's that sulfur from the S-CoA, and then this side of the molecule is esterified to two phosphates, which are esterified to-- I'm not going to draw it out, but this would have an adenine base and a phosphate there. So basically, this is ADP, with a phosphate added to the 3 prime position of the ADP molecule, added to pantothenic acid, added to this short chain with the sulfur on the end. And this whole molecule together is coenzyme A. And so when we say acetyl-CoA, it's this giant molecule, S-thioester to the carbonyl, the acid on acetate, to CH3. And so that would be, basically, acetyl-CoA. And so one convenient thing about this is it's much easier than drawing that big molecule, but it is a little bit misleading in terms of its size. So, that's coenzyme A. Next one I want to talk about is FAD which stands for flavin adenine dinucleotide. So, flavin adenine dinucleotide is an electron carrier, just like NAD plus. And it's derived from the vitamin riboflavin, also referred to as vitamin B2. Another thing from the side of your cereal box, and basically looks like this. So, like NAD, it's a dinucleotide. And so here's ADP, just like we do for CoA, or just like one end of NAD. One difference is that the other nucleotide down here actually isn't technically a sugar. It's ribitol instead of ribose. What does that mean? It doesn't have an aldehyde. Instead, it is just a five-carbon chain where all of the carbons are alcohols. And so since there's no aldehyde, it doesn't form a ring. And the base on this end, as a nicotinamide, is this flavin group, which looks like this. And so this is FAD, which is in the oxidized form. Turns out, in the oxidized form, FAD is yellow, hence riboflavin-"flavin", yellow. And the reason it's yellow-- you see there's a conjugated double bond here across this part. It turns out this is the active part of the molecule, and it works as follows. And so if you have a hydride ion, remember the way we can transfer two electrons. Can transfer the two electrons that way, and that allows it to generate. And I'll just draw the middle, here. Active part of the molecule. So, that would be these two nitrogens. We've added two electrons to it. And so this is abbreviated FADH2, or the reduced form of FAD. And it is colorless, because now you no longer have that conjugated double bond system, loses its color. And so you can follow whether FAD is oxidized or reduced by just looking at color change. So, that's FAD. It's an electron carrier, carries electrons by a hydride transfer, very similarly to what we described for NAD, but obviously a different molecule.

~15:13And the last molecule, the last co-factor that we need for this reaction, is lipoic acid. Which, unlike most things in metabolism, doesn't have an abbreviation and also functions as an electron carrier. And so lipoic acid looks like this. So, that is lipoic acid. This is in the oxidized form. And so where it's oxidized is here at this sulfur-sulfur bond. So, you can think of this as the same as a disulfide bond that you learned about from Professor Yaffe in proteins. And so this is the oxidized form of the disulfide bond. If I take this hydride, transfer two electrons across that bond, then it goes to-- and I'll just draw here the active end. Then we go here to the reduced form of lipoic acid, and there's no abbreviation for oxidized or reduced lipoic acid. It's just lipoic acid, oxidized lipoic acid, reduced. And how they're oxidized and reduced is basically very similar to the disulfide bonds that you saw in proteins being oxidized, disulfide bond being oxidized, or it can be reduced to not be a disulfide bond. Now, it turns out that these cofactors, our TPP, FAD, and lipoic acid, are all stably associated with different subunits of a multi-protein complex that assembles to catalyze that reaction to convert pyruvate to acetyl-CoA. The enzyme or enzyme complex that carries this out is referred to as PDH, sometimes abbreviated PDC, which stands for the pyruvate dehydrogenase complex. So, pyruvate dehydrogenase, PDH, pyruvate dehydrogenase complex, PDC. This complex is basically three different polypeptides that come together to form a complex and catalyze that reaction. Now, where this complex sits-- so, this is the mitochondria. This is the matrix. That's where this pyruvate dehydrogenase reaction occurs. That's where the TCA cycle occurs. And this PDH complex is basically sitting here at the membrane on the matrix side of the membrane. Now, the three different polypeptides that come together to form this complex are creatively named E1, E2, and E3, four. Enzyme one, two, and three. And each of these, as I alluded to, is associated with a different co-factor. And so E1 is associated with thymine pyrophosphate. E2 is associated with lipoic acid. And E3 is associated with FAD. Now, let's go through the mechanism for how this pyruvate dehydrogenase reaction works. So remember, this is the active part of TBP plus. It is bound in the active site of E1. And it reacts with pyruvate to catalyze alpha decarboxylation, just like we described for conversion of pyruvate to alcohol. So, you're going to see exactly the same mechanism that we drew before. So, that decarboxylates the alpha-keto acid, just like we saw to generate acetaldehyde. The difference is rather than resolve this such that this carbon has the same oxidation state and make acetaldehyde, instead the next step of this reaction is going to be oxidized by reducing lipoic acid. So, the active part of lipoic acid that is in the active site of the E2 subunit. So this will now regenerate E1, but what we're left with, then, is this. Now, this intermediate bound to E2. Here's where coenzyme A can come in, which will then generate acetyl-CoA. But now we are left with E2 in the reduced state, rather than being in the oxidized state. So E2 has to be re-oxidized in order for this complex to carry out the next catalytic cycle, and the way that works is as follows.

~18:56So you have FAD bound on E3. And so you have a hydride ion from the oxidation of E3 that can be transferred to FAD. That will generate FAD from the oxidized to the reduced form. So re-oxidize lipoic acid on E2, reduce FAD to FADH2, and then that FADH2 can be re-oxidized back to FAD via transferring those electrons to NAD plus to generate NADH. So in this case, FADH2 re-oxidized the FAD, NAD plus reduced to NADH. And so, effectively, what this happens is that enzyme E1 and E2 cooperate to call what's referred to as oxidative alpha decarboxylation of pyruvate, while adding -CoA, so that's where you get acetyl-CoA, with reduction of lipoic acid in E2, and then E3 re-oxidizes the lipoic acid in E2 while generating NADH. This NADH, once it's generated, of course those electrons have to go somewhere, so they, like in glycolysis-- it also needs to regenerate to NAD at some point. This is ultimately the electrons that end up on oxygen, and it's really this series of electron transfers, with oxygen being a good electron acceptor, that ultimately allows controlled energy release during carbon oxidation. And cells, you'll see, can use that to make ATP or do other kinds of work. So, the net reaction and/or another way to draw the pyruvate dehydrogenase reaction would be as follows, and that's taking pyruvate to acetyl-CoA. And so we're going to take coenzyme A and release CO2. This is done by TPP plus, as part of E1. That involves converting the lipoic acid in E2 from the oxidized to the reduced state. That lipoic acid then has to be re-oxidized. If something's oxidized, something else has to be reduced. That's FAD on E3, which also then cycles between the oxidized and reduce state. And ultimately, those electrons ended up being transferred to NAD plus to generate NADH. And so the PDH complex is basically a chain of electron transfer reactions. And it's the first example of a chain of electron transfer reactions. We're going to see that there is the electron transport chain in the mitochondria, effectively does the same thing. And by coupling oxidation and reduction reactions across chains of molecules like this, effectively as a preview, allows the stepwise energy release of these oxidation reactions to occur. So remember, if we burn glucose, completely oxidize it in one step, where those electrons are directly transferred to oxygen in combustion. Lots of energy released, but all in one step. By doing these stepwise electron transfers, we can then basically break up that energy release in a way that can be captured by cells to do work. Here, the way energy is captured, it's not so obvious in this electron transport reaction. But effectively, you're using oxidation of the ketone on pyruvate, with decarboxylation to the acid to generate, rather than just the acid, a thioester bond. And that's that thioester bond-- as well as NADH, that's energy as well-- but it's really that thioester bond that then can be recaptured later to drive synthesis of citrate in the TCA cycle. Now, next what I want to do is I want to discuss the TCA cycle reactions. Now that you see how you can get acetyl-CoA, at least from pyruvate, I want to discuss how you can now use that acetyl-CoA and oxidize it back to combine it with oxaloacetate, and oxidize it back to make citrate, and then oxidize it back to oxaloacetate to run the TCA cycle. OK, great. So, the first reaction of the TCA cycle. Here's acetyl-CoA. It will combine with oxaloacetate. This reaction is catalyzed by an enzyme called citrate synthase, and generates the six-carbon tricarboxylic acid citrate. So, how does this reaction work?

~22:58Here's drawing acetyl-CoA in a slightly different way. If I redraw this as the enol, this will generate citrate via, effectively, that mechanism. Next, citrate is converted by adding water across this carbon-carbon, or by removing water across this carboncarbon bond, to generate this intermediate called cis-aconitate. So this intermediate is called cis-aconitate. so all I did was dehydrate across that bond so there's a double bond there. And then if I re-add water across that bond, I generate this molecule called isocitrate. So effectively, to convert citrate to isocitrate, I'm moving the hydroxyl group from that carbon to this carbon. To do that, I basically dehydrate, make a double bond, remove water, readd water across that bond in the opposite direction to generate this molecule, isocitrate. This reaction is carried out by an enzyme called aconitase, and converts citrate to isocitrate. I think it's a little easier to see this reaction if I draw it a slightly different way. So, this here is just drawing citrate by just slightly rotating the molecule to look like that. And so I'm basically removing water here. And then I'm now just adding water back in the opposite orientation to generate isocitrate. Now, you'll notice when I drew this-- if you look at citrate, this is actually a symmetrical molecule, so the top half and the bottom half of citrate are identical. And so what's interesting about nature is that it treats these carbons- the green carbons that came from acetyl-CoA-- different from the side of the molecule that comes from oxaloacetate. And effectively, nature always moves the hydroxyl group to this carbon that came from oxaloacetate, and never moves it to this carbon that came from acetyl-CoA. This is an example where enzymes-- nature treats a symmetrical molecule like citrate in an asymmetrical way. And this has consequences for how carbon is actually traced through the entire TCA cycle. Because even though you might think things could get scrambled at citrate, they never do. Meaning an isocitrate-- it's always these green carbons that came from acetyl-CoA. You never get those green carbons on the other side of isocitrate. And so when we go through the TCA cycle, I'll keep these carbons green until the point where you can no longer distinguish which carbon came from came from which reaction. The next reaction is we're going to oxidize this carbon of isocitrate. So if we're going to oxidize that carbon, those electrons have to go somewhere. And so if we oxidize the carbon, we can use NAD plus as an electron acceptor, reduce it to NADH. That generates this intermediate. So hopefully this is clear to everybody at this point, but just in case. So, this carbon here and isocitrate. If I oxidize that alcohol to the ketone, now I generate a hydride ion. Those two electrons and the hydrogen can go to NAD plus and reduce it to NADH. That generates this intermediate. It's called oxalosuccinate. Which then, if you notice, the oxalosuccinate is now a beta keto acid. So alpha, beta. The acid group is beta to the ketone, so it's a beta keto acid. Remember, beta decarboxylation is favorable, and so I can lose that CO2. And what I'm left with is this molecule, which is called alpha keto glutarate. So this whole reaction here, the oxidation of the alcohol to the ketone, followed by beta decarboxylation of oxalosuccinate to alpha ketoglutarate, is carried out by an enzyme called isocitrate dehydrogenase. And of course, just to remind you, here's that beta keto acid in oxalosuccinate. And so that can decarboxylate, leading this enol. Which can, of course, rearrange back to the ketone that we see, an alpha ketoglutarate.

~26:57Now, if you look at alpha ketoglutarate, you'll notice that-- and I will redraw over here. So, this here is just me redrawing alpha ketoglutarate, which is often abbreviated alpha kG. Just drew it as a straight line. Now, if you'll notice, alpha keto glutarate is a alpha ketoacid. And so here the acid group is alpha to the ketone. So, an alpha keto acid. It's effectively just like pyruvate, but has this additional pieces on it. And it turns out the next step in the TCA cycle is the exact reaction that we saw with pyruvate dehydrogenase. It's alpha decarboxylation, oxidative alpha keto acid decarboxylation, as follows. So, we carry out. This is a molecule called succinyl-CoA. And so, you'll see what happened there is decarboxylated here, this carbon, while oxidizing this ketone to the acid and adding -CoA. That's a redox reaction. So NAD, NADH, it turns out this is exactly the same mechanism-- so I don't need to draw it again-- that I just showed you for pyruvate dehydrogenase. So it needs all the same cofactors-- TPP plus, lipoic acid, FAD. And in fact, it even shares some of the same enzyme complexes, subunits, as pyruvate dehydrogenase. And so this is a reaction that's catalyzed by alpha ketoglutarate dehydrogenase. Like pyruvate dehydrogenase, this is a complex, and so it has a unique E1, which makes sense. Remember, E1 of pyruvate dehydrogenase was actually the subunit that bound the pyruvate. E1 of alpha ketoglutarate dehydrogenase is unique. It binds alpha ketoglutarate instead of pyruvate. However, they share the same E2 and E3s, and so the E2s and E3s would carry out exactly the same reaction and play the same part in the mechanism of how you do this alpha oxidative alpha decarboxylation to turn alpha ketoglutarate into succinyl-CoA. Now, remember in pyruvate dehydrogenase, once we got that acetyl-CoA, we then use this CoA group to drive condensation with citrate. Well, in this case, what happens is you don't want to combine condensation here. Instead, what is going to happen is you want to use this CoA group to now generate ATP. And so this is going to couple release of the CoA to generate an ATP equivalent. It's actually GTP that's generated by the TCA cycle. And so, this is going to generate this molecule, succinate. And the enzyme that does this is called succinic bio-kinase. So, let's go through over here how this enzyme works. So here's succinyl-CoA, basically using favorable loss of the CoA breaking the thioester bond to generate this acid anhydride. We saw an acid anhydride before in glycolysis. Remember, we made 1-3-bisphosphoglycerate, so that's a good phosphate donor. And then that can be used to generate succinate, and transferring that phosphate to GDP to make GTP, just like 1-3bisphosphoglycerate, was able to transfer the phosphate from the acid anhydride to ADP to make ATP in glycolysis. So, the next step is to oxidize this carbon-carbon bond in succinate. So if we're going to oxidize a carbon-carbon bond, those electrons have to go somewhere. So there's our carbon-carbon bond. And so if we generate hydride ion just like we did in other oxidation reactions, but this hydride ion is transferred not to NAD, but instead to FAD, a different electron carrier, to generate if FADH2. Now, I should point out succinate, like citrate, is a symmetrical molecule. But at this point, nature doesn't tell the difference. Once it generates succinate, this molecule now gets scrambled. And so everything downstream of succinate, you no longer know which carbons came from acetyl-CoA. This generates this molecule called fumarate, and this reaction is carried out by an enzyme called succinate dehydrogenase, often abbreviated SDH for succinate dehydrogenase.

~30:56Now, the next reaction is, we're going to add water across this double bond of fumarate. And that generates this intermediate, malate. This reaction is carried out by an enzyme called fumarate hydratase. And it's simply adding a water molecule across that double bond. Once we have malate, if you look what's the difference between malate and oxaloacetate, the difference is that in oxaloacetate, this carbon is a ketone. Whereas in malate, it's an alcohol. And so if we want to turn this carbon from the alcohol into the ketone, that is, of course, a oxidation reaction, and so those electrons have to go somewhere. Don't need to draw the mechanism again. It's basically just the hydride transfer to oxidize this to an alcohol, to the ketone. That oxidation couples to a reduction of NAD plus to NADH. This is carried out by an enzyme called malate dehydrogenase. And doing this completes the TCA cycle, regenerating oxaloacetate, that can then recombine with another acetyl-CoA to go through another round of the cycle. You'll notice that going through this cycle, there's two CO2s lost. One of them is lost here at the isocitrate to alpha ketoglutarate reaction. So, this decarboxylation from oxaloacetate to alpha ketoglutarate. That beta decarboxylation, that's the first CO2. The other one is lost here at the alpha ketoglutarate. The alpha ketoglutarate dehydrogenase step, where you have this alpha decarboxylation to take alpha ketoglutarate to succinyl-CoA oxidative alpha decarboxylation. Now, what's cool about this, as you noticed, we just discussed all the reactions of the TCA cycle. And I showed you, reminded you, of some chemistry that you've already seen, but unless you count the chemistry we showed you earlier for how the PDH and alpha ketoglutarate dehydrogenase reactions work with E1, E2 and E3, with lipoic acid, FAD. That was obviously new for today. But other than that, everything else was chemistry that you've already seen. And this really points out the point that I made earlier, that metabolism is really variations on relatively few reactions. We've just repurposed some of the same tricks, if you will, that we're used in glycolysis, and allowed it to now do an entire different pathway, the TCA cycle. It also points out how Hans Krebs was-- well, is still-remarkable, able to figure out from chemistry alone, because there's actually quite a bit of logic to the way metabolism works. Now, I want to say this again. Note there were two carbons that entered acetyl-CoA, and two carbons that were lost to CO2. But if you look, the green carbons remain in the same places until they get to succinate. and so the two carbons that enter are not lost on the first turn of the cycle. It's actually two carbons that came from oxaloacetate that are converted to CO2 as that acetyl-CoA goes through the cycle. And so to oxidize the exact carbons from acetyl-CoA to CO2 requires more than one turn of the cycle.

~34:07You'll also notice that the cycle is oxidation. And so oxidation reactions, of course, release energy. We've talked about that. And so it's favorable. And the products, if you will, are three NADH molecules. You can say plus one more NADH if we're going all the way from glucose or from pyruvate. Glucose derived pyruvate because the pyruvate dehydrogenase reaction also generates an NADH to make that a acetyl-CoA. One FADH2, as well as one GTP molecule. And so lots of oxidation going on here. We completely oxidized two carbons to CO2, so that's energy release. But you notice you only get one GTP from the molecule.

~34:49Now, this GTP, of course-- that reaction, the succinic thiokinase reaction, like the reactions we saw on glycolysis, is such that it can generate GTP at a high DGP-GDP ratio, or ATP-ADP ratio. Remember, those are equivalent. Those energy charge are similar, and so that makes sense. But most of the energy released is actually reducing NAD and FAD to NADH and FADH2. And of course, these need to transfer their electrons somewhere else, and that's the role of oxygen. Oxygen, remember, is a very good electron donor, and so it's the ultimate transfer of those electrons from these molecules to oxygen that also provide energy that the cell can use to do work, but it does so, in a way, by charging up different ratios in the cell. So the NAD-NADH or the FADH2-FAD ratios. And just like we talked about, the ratio or the energy of ATP is in the interconversion between ATP and ADP. It's the ratio that drives the free energy change. The same thing exists for an NADH and NAD, FADH2 and FAD. And so charging up these ratios while passing through the TCA cycle, and the ultimate downstream transfer of those electrons to oxygen, really is where most of the energy is captured as carbon is oxidized through the TCA cycle. And exactly how that works and how it can be related to ATP will be something that'll be more explicit in the coming lectures. Now, I want to point out apart from the oxidation, there's actually lots of intermediates made here. And it turns out, a bunch of these intermediates are useful for cells to make stuff. So we talked about gluconeogenesis. Gluconeogenesis needs electron balance. We get NADH from the TCA cycle, and so you can think of gluconeogenesis as an alternative to fermentation to dispose of electrons. Well, you can use the NADH from the TCA cycle to run gluconeogenesis as well. But beyond the cofactors, the carbon itself. So citrate, I've alluded to now a few times, is important as a precursor to make fat. We'll discuss that in later lectures, too. But other intermediates in this pathway are useful for various amino acids and nucleic acids. And so there's lots of things that can come from the TCA cycle that cells can find useful to do, not just catabolism, but also anabolic processes. Now, the way the TCA cycle works, though, is that there's actually an issue if you want to use the intermediates from the TCA cycle to make stuff. And so what is that issue? Well, the TCA cycle functions at a site as a cycle. And so, if we're going to take things in and out of it, that has consequences for how the cycle runs. Now there's a couple words for this that I want to just introduce to you. The first one is cataplerosis and the second one is anaplerosis. And so cataplerosis is the act of removing stuff from a metabolic cycle. So, we're going to remove citrate from the cycle to make fat. That's cataplerosis. And anaplerosis is adding stuff back to a metabolic cycle so it can continue to function. Viewing this is really evident if you think of the TCA cycle as a chicken and egg problem. So, the very first time acetyl-CoA was generated, how do you start the TCA cycle in the first place? You can't add it to the TCA cycle unless you have oxaloacetate to combine with the acetyl-CoA, which can then generate another oxaloacetate. So where does the first oxaloacetate come from? Well, we already talked about one reaction. We talked about it in the context of gluconeogenesis. That can solve this problem.

~38:47And so we have pyruvate. And so we talked earlier today how we can do oxidative decarboxylation of pyruvate to give acetyl-CoA. But we talked to in the gluconeogenesis lecture how we can add a CO2 pyruvate to generate oxaloacetate. So if I do those two reactions, now I have all the carbon I need to generate a citrate and start off the TCA cycle. Now obviously, if I do cataplerosis and I remove that citrate I made to make fat, well, now I need two pyruvate again to generate the next citrate if I'm using this pathway, because every time I bring an acetyl-CoA into the cycle, I need an oxaloacetate to combine it with. And so if I remove something, I have to add something back. And so pyruvate to oxaloacetate, the pyruvate carboxylase reaction is an example of an anaplerotic reaction. Now what this means, though, is that in order to do an anaplerosis, you have to be able to generate four-carbon oxaloacetate, or a four-carbon molecule. Now, pyruvate carboxylase, pyruvate to oxaloacetate, allows you to do that. We can take a three-carbon molecule and generate four-carbon oxaloacetate. However, if we start from a two-carbon molecule like acetate or acetyl-CoA that enters the cycle, it's actually not so simple to take that two-carbon molecule and turn it into four-carbon oxaloacetate. And in fact, humans lack any enzymes that allow them to take a two-carbon unit, to take acetate or acetyl-CoA, and turn it into anything that's longer than net-- turn it into anything that's longer than two carbons. And this has important implications for human physiology, because what it says is that we can't make glucose from anything that starts with something less than three carbons long. So if you drink alcohol and you metabolize that alcohol to acetate, it turns out you take fat and you break down fat also to acetyl-CoA, to acetate two carbons long. There is no way to turn those molecules into glucose, because you cannot generate the oxaloacetate to do the anaplerosis that's necessary to get it there. What this means is that our body can only store calories that come from two carbon units, fat or alcohol, as fat. We can never turn them back into glucose or make glycogen. And this is very relevant for those of you that go to medical school, because it's relevant to our physiology. And that is, when our bodies exhaust all of our stores of glucose, what happens? Our liver can no longer do gluconeogenesis. And so what happens? Now it has to switch over to doing something else. It has to work with two carbon units. And ultimately, this is ketone metabolism, which we'll talk about in a few lectures. It also said that the body-- that this is also the basis of a very old adage that's out there that some of you may have heard-- that you need to have some other fuel if you're going to burn fat. The basis for that is that fat is turned into two carbon units, acetyl-CoA units. And so if you're going to take those acetyl-CoA units and ultimately burn them away, turn them into CO2, you need a source of oxaloacetate, or your TCA cycle won't work. And you don't need a lot of something, but it is true. You can't start just with acetyl-CoA as a human and turn it into CO2, so you need some-- at least a little bit of oxaloacetate to get your TCA cycle started. Now, that's a problem that we as humans and other mammals face, but it turns out there's lots of microbes out there that grow just fine on acetate or on alcohol, even if it's only carbon source. And so those organisms must have some way to build stuff from two carbon units. That is a way to use two carbon units and do an anaplerosis. And it turns out the way they do this is via something called the glyoxylate cycle.

~43:06And so the glyoxylate cycle is an alternative version of the TCA cycle that effectively uses two enzymes that we lack as mammals. And so I'll quickly tell you about it here. So, this is isocitrate from the TCA cycle. And some microbes have an enzyme called isocitrate lyase. And what isocitrate lyase does is basically splits citrate in half, such that the top portion of the molecule is another TCA cycle intermediate, succinate. And the bottom portion of a molecule is this two carbon aldehyde called glyoxylate. Glyoxylate can react with acetyl-CoA by another enzyme that we lack as humans called malate synthase. And I don't have time to show the mechanism again, but malate synthase basically adds the two carbons from acetyl-CoA to the aldehyde, the carbonyl, the aldehyde carbonyl of glyoxylate in a reaction that is, for all intents and purposes, exactly what happens in citrate synthase. That will generate this molecule, which is malate, also in the TCA cycle. And so having these two extra reactions, isocitrate lyase and malate synthase, gives microbes the ability to have acetyl-CoA be anaplerotic. And so how does that work? Well, that's because if we start with one oxaloacetate, four carbons, and acetyl-CoA, two carbons, can run the TCA cycle and make citrate, six carbons. Turn that citrate into isocitrate. Use isocitrate lyase to generate glyoxylate, two carbons. Plus succinate. That succinate can run through succinate dehydrogenase to generate malate, which you can go through malate dehydrogenase to generate oxaloacetate. This glyoxylate can start with a second acetyl-CoA. Two carbons come together, generate malate. That generates a second malate molecule, which can then exit the cycle as malate or oxaloacetate or whatever you want. And so basically, it allows two acetyl-CoAs to net generate an oxaloacetate, and so net generates a way to do an anaplerosis from two carbon units by having this malate synthase reaction and this isocitrate lyase reaction and run this alternative version of the TCA cycle, called the glyoxylate cycle. And it's a nice way how life-- again, no new chemistry here, just variations on what we've already shown-- repurposed the similar chemistries that it's already using as a way to live off of carbon sources that contain only two carbons, like ethanol or acetate. So in closing today, the last thing I want to talk about, very briefly, is how the TCA cycle is regulated. And we don't need to spend a ton of time on this, because it really follows principles that make sense, particularly when we think about things that we've already talk about. And so, regulation of the TCA cycles is of course important, and it's really a critical hub, both for anabolic and catabolic pathways. So, you needed to get energy to fully oxidize carbon, but it's also a useful place to get stuff. And before I talk about regulation, I just want to point out that many of the enzymes in the TCA cycle, even though we talk about it in the mitochondria and we're about to talk about regulation in terms of catabolism-- that is oxidation, ways to release energy-- many of these enzymes are also in other locations in the cell, because there's functions for them in making stuff that is very different than what goes on in the TCA cycle. And the regulation that I'll tell you about, and that comes up on MCAT exams and stuff like that, usually talks about this pathway as a catabolic pathway, as a way to make CO2, to make ATP. However, recognize that there's also variations on this pathway that can use in anabolic things, making stuff. And that regulation is something that really is not necessarily what we're going to talk about here and is a little bit less well-understood.

~47:10But at least the regulation, in terms of catabolism, if we put it in context of glucose metabolism-- so here's glycolysis, turning glucose into pyruvate. And then that pyruvate can operate through the pyruvate dehydrogenase reaction to generate acetyl-CoA. That acetyl-CoA can combine with oxaloacetate to generate citrate. That citrate, of course, can be used to generate fat, as we've talked about. That can go to isocitrate. Isocitrate to alpha ketoglutarate. That's catalyzed by isocitrate dehydrogenase, which I will abbreviate IDH. Alpha ketoglutarate to succinate. More correctly, to succinyl-CoA by alpha ketoglutarate dehydrogenase, Alpha KGDH. And then this back to oxaloacetate. And so the main enzymes that are typically discussed as being regulated are alpha ketoglutarate dehydrogenase, isocitrate dehydrogenase, and pyruvate dehydrogenase. I also drew glycolysis. And I'll just write over here, gluconeogenesis up here, because we can now see more fully how this plays in with citrate acting as a positive regulator with gluconeogenesis and an inhibitor of glycolysis. Again, we're full in citrate, stop running glycolysis, start running gluconeogenesis. Now, the other regulation. If you have a lot of acetyl-CoA, stop making it from pyruvate. acetyl-CoA is a negative regulator of PDH. This is carbon oxidation. Releases a lot of energy, generates a lot of ATP, generates a lot of NADH. If you have lots of those things, no reason to keep sending carbon into acetyl-CoA to go into the TCA cycle. Ultimately, we have to send those electrons somewhere. So if there's nowhere to put them, low oxygen also inhibits pyruvate dehydrogenase. And of course, if you need more energy, if ADP is high, that activates pyruvate dehydrogenase. So at least in terms of glucose, complete glucose oxidation, a lot of regulation happens at pyruvate dehydrogenase. And a lot of it really makes sense. High levels of ATP, high levels of NADH, also inhibit isocitrate dehydrogenase and alpha ketoglutarate dehydrogenase. Makes sense. Succinol-CoA high, inhibit alpha ketoglutarate dehydrogenase. High levels of ADP, need to release more energy, activate alpha ketoglutarate dehydrogenase. Again, these are the feedbacks that people talk about on board exams. Good to know, but you can almost guess what they would be from first principles. Because remember, this is a pathway that releases a lot of energy. High energy high ATP, high NADH-- don't run the cycle, don't enter carbon in the cycle. Low energy, high ADP, put carbon in the cycle, run the cycle faster. Makes sense. Also makes sense of the reciprocal regulation of glycolysis and gluconeogenesis. Great. Next time, we will talk more about how we can oxidize fatty acids, fat, by accessing acetyl-CoA and entering it into the cycle. Thanks.

3.4柠檬酸循环（TCA Cycle）八步反应全解析：三羧酸的化学重排与四次脱氢

一、Hans Krebs 的天才拼图与柠檬酸循环全景

当丙酮酸经 PDH 复合体变成二碳的乙酰-CoA（或者脂肪酸 β-氧化、乙醇代谢、氨基酸降解产生乙酰-CoA）后，细胞如何把这剩下的两个碳原子彻底氧化成 CO₂？如果直接去氧化乙酸（CH_3COOH）的甲基碳，在温和的生理温度下几乎无法打断其稳定的 C-C 键。1937 年，德国生物化学家 Hans Krebs 凭借惊人的化学直觉解开了这个谜团：细胞不是直接硬啃二碳乙酸，而是先让二碳乙酰-CoA 与四碳的草酰乙酸（OAA）缩合成六碳的柠檬酸（Citrate，一种三羧酸），再通过一整套八步环状反应，分两次优雅地脱去两分子 CO₂（β-脱羧与 α-脱羧），并在第八步重新再生出四碳草酰乙酸！这就是著名的 柠檬酸循环（Citric Acid Cycle），又称三羧酸循环（Tricarboxylic Acid Cycle, TCA 循环）或 Krebs 循环 2014/2018·名解。

二、柠檬酸循环八步反应的有机化学机理、限速酶与抑制剂逐一拆解

正如 Vander Heiden 教授在 L23 课堂上感叹的：“TCA 循环没有发明任何古怪的新化学，它只是把我们在糖酵解和 PDH 中见过的羟醛缩合、脱水/水合、醇氧化、β-脱羧、α-氧化脱羧和硫酯键能量偶联，像交响乐一样重新编排在了一起！”让我们逐一走过这八步反应：

第 1 步（缩合 · 限速步 1）：乙酰-CoA 与草酰乙酸缩合生成柠檬酸

乙酰-CoA（2C） + 草酰乙酸（OAA，4C） + H_2O ──(柠檬酸合酶（Citrate synthase）)──► 柠檬酸（Citrate，6C） + CoA-SH + H⁺ (Δ G'^° = -32.2 kJ/mol)

• 催化机理（克莱森酯缩合与硫酯水解驱动）：在柠檬酸合酶活性中心（His274、His320 与 Asp375 协同酸碱催化），乙酰-CoA 的甲基首先脱去一个质子形成亲核性的烯醇负离子（碳负离子），进攻草酰乙酸的 α-酮羰基碳，生成酶结合态的中间物——柠檬酰-CoA（Citryl-CoA）。紧接着，酶引入第 1 分子 H_2O，立即将柠檬酰-CoA 的高能硫酯键水解（释放高达 -31.4 kJ/mol 的自由能！），生成柠檬酸和游离 CoA-SH。
• 为什么柠檬酸合酶必须“先结合草酰乙酸，后结合乙酰-CoA”（诱导契合机制）？ 既然柠檬酸合酶专门催化高能硫酯键水解，万一乙酰-CoA 先钻进活性中心，岂不是要被水分子直接白白水解成乙酸和 CoA-SH（浪费高能硫酯键）？自然界用教科书级的诱导契合（Induced fit）解决了这一难题：游离的柠檬酸合酶处于开放构象，根本没有乙酰-CoA 的结合位点！只有当底物草酰乙酸（OAA）率先结合到酶上时，才会诱导酶的两个结构域发生大幅度闭合，从而凭空创造出乙酰-CoA 的结合口袋，确保乙酰-CoA 只有在缩合变成柠檬酰-CoA 之后才会被水解！
• 命名辨析：为什么叫“柠檬酸合酶（Synthase）”而不叫“合成酶（Synthetase）”？ 因为反应直接靠乙酰-CoA 自身的高能硫酯键水解供能，没有 ATP 或 GTP 等核苷三磷酸直接参与！

第 2 步（脱水与再水合异构）：柠檬酸经顺乌头酸异构为异柠檬酸

柠檬酸（Citrate，叔醇） ↔(顺乌头酸酶（含 [4Fe-4S]）｜-H_2O) [顺乌头酸（cis-Aconitate）] ↔(顺乌头酸酶｜+H_2O) 异柠檬酸（Isocitrate，仲醇） (Δ G'^° = +13.3 kJ/mol)

• 为什么柠檬酸不能直接脱氢氧化，非要先异构为异柠檬酸？ 请看柠檬酸 C3 位的羟基：它连接在一个没有氢原子的季碳上，属于叔醇（Tertiary alcohol）！在有机化学中，叔醇如果不打断碳-碳键，根本无法被氧化成酮羰基！因此，细胞必须派出顺乌头酸酶（Aconitase，又称乌头酸酶），先把 C3 位的羟基“搬家”到相邻的 C2 位上——先脱去一分子 H_2O 在 C2=C3 间形成双键，生成不脱离酶的中间物顺乌头酸（cis-Aconitate）；随后再把水分子以相反的取向加回双键上，生成 C2 位带羟基的异柠檬酸（Isocitrate，仲醇 Secondary alcohol），为下一步氧化成酮铺平道路！
• 铁硫簇（[4Fe-4S]）与柠檬酸发酵工业应用：顺乌头酸酶活性中心含有一个非血红素的 [4Fe-4S] 铁硫簇，其中的一个铁原子直接配位结合底物的羧基与羟基以催化脱水与水合。在工业生产柠檬酸（黒曲霉发酵）时，向培养基中加入铁螯合剂（如亚铁氰化钾）去除铁离子，顺乌头酸酶即失活，碳流便源源不断地停留在柠檬酸并大量分泌积累！
• 毒理学经典——氟乙酸的“致死性合成（Lethal Synthesis）”：氟乙酸（Fluoroacetate，剧毒灭鼠药“1080”及南非毒鼠子植物毒素）本身对任何酶都无抑制作用；但进入细胞后，它会被当作乙酸活化为氟乙酰-CoA，进而被柠檬酸合酶与草酰乙酸缩合生成氟柠檬酸（Fluorocitrate）；氟柠檬酸是顺乌头酸酶的超强抑制剂，能死死卡住顺乌头酸酶活性中心，使 TCA 循环彻底中断而致死！这种把无毒前体通过细胞自身代谢酶转化为剧毒抑制剂的过程，称为致死性合成。

第 3 步（第 1 次氧化脱羧 · 限速步 2）：异柠檬酸氧化脱羧生成 α-酮戊二酸

异柠檬酸（6C） + NAD⁺ ──(异柠檬酸脱氢酶（IDH，Mg²⁺/Mn²⁺）)──► [草酰琥珀酸] ⟶ α-酮戊二酸（α-KG，5C） + CO₂↑ + NADH + H⁺ (Δ G'^° = -20.9 kJ/mol)

• 两步化学机理（醇氧化 + β-酮酸脱羧）：异柠檬酸变成仲醇后，立即被异柠檬酸脱氢酶（IDH）催化分两步反应：① 氧化脱氢：NAD⁺ 夺取异柠檬酸 C2 上的氢负离子，将 C2 仲醇氧化为酮羰基，生成第 1 分子 NADH 和不离开酶的中间体——草酰琥珀酸（Oxalosuccinate）；② β-脱羧：Vander Heiden 教授在黑板上标出，草酰琥珀酸的 C2 酮基相对于 C3 上连接的羧基恰好处于 β-位！作为一个不稳定的 β-酮酸，它在酶活性中心 Mn²⁺（或 Mg²⁺）螯合稳定烯醇负离子的协助下，极易发生自发 β-脱羧，释放出 TCA 循环的第 1 分子 CO₂，生成五碳的 α-酮戊二酸（α-Ketoglutarate, α-KG）。
• 同工酶辨析：哺乳动物细胞中有两类异柠檬酸脱氢酶——参与 TCA 循环的是仅位于线粒体基质的 NAD⁺ 依赖型 IDH（受 ADP 和 Ca²⁺ 别构激活，受 ATP 和 NADH 抑制）；此外在线粒体和细胞质中还存在 NADP⁺ 依赖型 IDH（主要用于产生 NADPH 供生物合成与抗氧化，近年肿瘤代谢研究发现 IDH1/IDH2 突变会将 α-KG 异常还原为致癌代谢物 2-羟基戊二酸 2-HG）。

第 4 步（第 2 次氧化脱羧 · 限速步 3）：α-酮戊二酸氧化脱羧生成琥珀酰-CoA

α-酮戊二酸（5C） + CoA-SH + NAD⁺ ──(α-酮戊二酸脱氢酶复合体（3 酶 5 辅酶）)──► 琥珀酰-CoA（4C） + CO₂↑ + NADH + H⁺ (Δ G'^° = -33.5 kJ/mol)

• 与 PDH 复合体的“孪生兄弟”关系：请看 α-酮戊二酸的结构——它跟丙酮酸一样，也是一个典型的 α-酮酸（只是比丙酮酸多了一个 -CH_2COO^- 尾巴）！因此，α-酮戊二酸脱氢酶复合体（α-KGDH）的组织结构、催化机理与丙酮酸脱氢酶复合体（PDH）完全同源：同样由 3 种酶（E1 α-酮戊二酸脱氢酶、E2 二氢硫辛酰转琥珀酰酶、E3 二氢硫辛酰脱氢酶）和完全相同的 5 种辅因子（TPP、硫辛酸、CoA-SH、FAD、NAD⁺）组成 2024·单选！甚至它们连第 3 个亚基 E3 都是由同一个基因编码的完全相同的多肽链（只是 E1 和 E2 因识别底物不同而各具专一性）！
• 这步反应高度放能且不可逆，释放出 TCA 循环的第 2 分子 CO₂ 和第 2 分子 NADH，并将氧化释放的能量储存在琥珀酰-CoA（Succinyl-CoA）的高能硫酯键中（Δ G'^° ≈ -36 kJ/mol）。
• 与 PDH 的唯一调控区别：α-KGDH 同样受产物琥珀酰-CoA、NADH 抑制，受 Ca²⁺ 和 ADP 激活，且同样被亚砷酸盐抑制；但 α-KGDH 的 E1 亚基不受蛋白激酶/磷酸酶的共价磷酸化修饰调节！

第 5 步（唯一底物水平磷酸化）：琥珀酰-CoA 转化为琥珀酸并合成 GTP（ATP）

琥珀酰-CoA + GDP（或 ADP） + P_i ↔(琥珀酰-CoA 合成酶（琥珀酸硫激酶）) 琥珀酸（Succinate） + GTP（或 ATP） + CoA-SH (Δ G'^° = -2.9 kJ/mol)

• 这是 TCA 循环中唯一一步底物水平磷酸化反应（直接生成高能磷酸键，无需氧气和呼吸链参与） 2010·判断2013/2014/2016·单选！
• 三步磷酸基传递机理：在第 1 步柠檬酸合酶中，高能硫酯键水解的能量被用来推动碳-碳键缩合；而在第 5 步琥珀酰-CoA 合成酶（Succinyl-CoA synthetase，又称琥珀酸硫激酶 Succinic thiokinase）中，细胞将琥珀酰-CoA 的高能硫酯键兑现成了 1 分子 GTP（或 ATP）：

1. 无机磷酸 P_i 亲核进攻琥珀酰-CoA，置换出 CoA-SH，生成与酶结合的高能酰基磷酸酸酐——琥珀酰磷酸（Succinyl-phosphate，类似于糖酵解中的 1,3-BPG）；
2. 酶活性中心的一个组氨酸（His）残基夺取琥珀酰磷酸上的磷酸基，释放出游离的琥珀酸（Succinate），自身变成高能的磷酸化组氨酸（3-Phosphohistidine）中间体；
3. 磷酸化 His 摆向核苷二磷酸结合位点，将磷酸基直接转移给 GDP（或 ADP），生成 GTP（或 ATP）！

• 两个高频必考细节：① 物种差异：高等动物拥有分别专一于 GDP（主要在肝、肾等合成代谢活跃组织）和 ADP（主要在骨骼肌、心肌等耗能组织）的两种琥珀酰-CoA 合成酶同工酶，教材标准总反应式统一写 GTP；而植物和细菌的该酶则直接以 ADP 为底物生成 ATP！生成的 GTP 与 ATP 可通过核苷二磷酸激酶（GTP + ADP ↔ GDP + ATP，Δ G'^° = 0）自由等能互换。② 命名陷阱：为什么这步反应明明在分解琥珀酰-CoA，酶却叫“琥珀酰-CoA 合成酶（Synthetase）”？因为该反应近平衡可逆（Δ G'^° = -2.9 kJ/mol），酶是按照逆反应（消耗 GTP/ATP 合成琥珀酰-CoA）来命名的（凡叫“合成酶 Synthetase”者必有 NTP 参与）！

第 6 步（第 3 次脱氢 · 唯一内膜酶）：琥珀酸脱氢生成延胡索酸

琥珀酸（Succinate） + E-FAD ↔(琥珀酸脱氢酶（呼吸链复合体 II）) 延胡索酸（Fumarate，反丁烯二酸） + E-FADH₂ (Δ G'^° = 0 kJ/mol)

从第 6 步到第 8 步，四碳二羧酸已經完成了脱羧使命，接下来的任务是把对称的琥珀酸（丁二酸）一步步氧化改造成不对称的 α-酮酸——草酰乙酸（其反应序列为：烷烃脱氢成烯烃 → 双键加水成仲醇 → 仲醇脱氢成酮，注意这套“脱氢-加水-再脱氢”的三步曲在下一章脂肪酸 β-氧化中将原封不动地重现！）。

• 为什么琥珀酸脱氢不用 NAD⁺，而必须以 FAD 为受氢体？ 因为将饱和烷烃链（-CH_2-CH2-）脱氢成碳碳双键（-CH=CH-）的还原电位较高（E'^° = +0.031 V），其释放的自由能根本不足以还原低电位的 NAD⁺（E'^° = -0.320 V），只能将电子交给电位较高的共价结合 FAD 2007·单选！
• 华农必考的“三大独一无二特征”：

1. 琥珀酸脱氢酶（SDH）是 TCA 循环中唯一镶嵌在线粒体内膜上的酶（其余 7 种酶均游离在线粒体基质中），它本身就是电子传递链的复合体 II（琥珀酸-泛醌还原酶） 2010/2018·填空！
2. 其辅基 FAD 通过异咯嗪环 C8α 甲基共价连接在酶蛋白的组氨酸（His）残基上，脱氢生成的 FADH₂ 不脱离酶，直接通过酶内的 3 个铁硫中心（Fe-S）将电子传给线粒体内膜中的泛醌（UQ → UQH₂），每分子 FADH₂ 彻底氧化产生 1.5 ATP（旧口径 2 ATP）。
3. 严格的立体专一性与丙二酸竞争性抑制：该酶只催化反式消除，专一生成反丁烯二酸（延胡索酸，Fumarate），绝不生成顺丁烯二酸（马来酸，Maleate）。三碳二羧酸——丙二酸（Malonate，^-OOC-CH_2-COO^-）因分子结构与底物琥珀酸（^-OOC-CH_2-CH2-COO^-）高度相似，能竞争性结合琥珀酸脱氢酶的活性中心，但由于少了一个亚甲基无法脱氢，是酶学史上最经典的竞争性抑制剂（Competitive inhibitor） 2009·单选！（Hans Krebs 当年正是利用丙二酸阻断实验证明了 TCA 循环的闭环本质！）

第 7 步（反式水合）：延胡索酸加水生成 L-苹果酸

延胡索酸（Fumarate） + H_2O ↔(延胡索酸酶（Fumarase）) L-苹果酸（L-Malate） (Δ G'^° = -3.8 kJ/mol)

• 催化特征：由延胡索酸酶（又称延胡索酸水合酶）催化，引入 TCA 循环的第 2 分子 H_2O（第 1 分子水在柠檬酸合酶处消耗）。该酶具有绝对的立体异构专一性：只能识别反式双键的延胡索酸（对顺式的马来酸完全无作用），且水分子的 OH^- 和 H⁺ 按反式加成，100% 专一生成 L-苹果酸（绝不生成 D-苹果酸）。

第 8 步（第 4 次脱氢 · 再生草酰乙酸）：L-苹果酸氧化再生草酰乙酸

L-苹果酸（L-Malate） + NAD⁺ ↔(苹果酸脱氢酶（MDH）) 草酰乙酸（OAA） + NADH + H⁺ (Δ G'^° = +29.7 kJ/mol)

• 热力学逆风翻盘的典范：最后一棒由线粒体苹果酸脱氢酶（MDH）将 L-苹果酸的 C2 仲醇氧化回酮羰基，生成 TCA 循环的第 3 分子 NADH，同时再生出四碳载体草酰乙酸（OAA），完成整个循环的闭环！
• 为什么苹果酸脱氢酶反应的标准自由能变高达 Δ G'^° = +29.7 kJ/mol（平衡极度偏向苹果酸一侧，标准状态下根本不能正向进行），但在活细胞线粒体内却能源源不断地向右生成草酰乙酸？
  答案就在紧随其后的第 1 步柠檬酸合酶反应（Δ G'^° = -32.2 kJ/mol）！因为柠檬酸合酶通过水解柠檬酰-CoA 的高能硫酯键，以极强的放能驱动力将刚生成的草酰乙酸瞬间抽走，使线粒体基质中草酰乙酸的稳态生理浓度被压低到惊人的 < 10^-6 mol/L（微摩尔级以下）！根据实际自由能公式：
  Δ G = Δ G'^° + RT ln ([OAA][NADH])/([L-Malate][NAD⁺])
  当分子中的 [OAA] 极度微小时，对数项 RTln Q 变成一个绝对值大于 29.7 kJ/mol 的大负数，从而将实际生理自由能变拉成 Δ G < 0，推动苹果酸源源不断地氧化为草酰乙酸！

三、柠檬酸循环的总反应式、三大限速酶与钙/能荷调控

把上述八步反应叠加起来，每进入 1 分子乙酰-CoA，运转一轮柠檬酸循环的总反应式为：

乙酰-CoA + 3 NAD⁺ + FAD + GDP（或 ADP） + P_i + 2 H_2O ⟶ 2 CO₂↑ + 3 NADH + 3 H⁺ + FADH₂ + GTP（或 ATP） + CoA-SH

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🎧 MIT 7.05 课堂原声英文实录对照（本节关联 2 讲 · 完整逐段实录）点击展开 / 收起 ▾

🎬 MIT 7.05 Lec 22 · Gluconeogenesis, Glycogen & TCA Cycle I⏱ ~50:00

~00:00MITOCW | 22. Glucogenesis/Carbohydrate Storage/TCA Cycle I

~00:02PROFESSOR:

~00:02So last time, we discussed glycolysis as a pathway. And I just want to remind everybody that, like all pathways, glycolysis has to be favorable. That is, delta G across the entire pathway has to be less than 0. We spent a lot of time discussing the regulation of glycolysis. And I just want to revisit this quickly. And remember, we discussed that the steps that are regulated are those with the biggest, quote, unquote, bio-energetic cost. That is, the biggest drop in delta G is as molecules would proceed across the pathway. And those are shown here-- the hexokinase step, the phosphofructokinase step, and the pyruvate kinase step of the pathway. Now this is largely true of most pathways that we will consider, in that these regulated steps are often those with the biggest bio-energetic cost. And the reason for that is that it makes a lot of sense. Once you go through those steps, it's very difficult to go back.

~00:55Also, you'll note that these are the steps that regulate entrance and exit from the pathway. And this also is something we'll commonly see and other pathways and also makes some sense. Just to remind everybody, I drew up here a skeletonized version of glycolysis and the most important regulatory features of it. Just to remind people that initially, hexokinase is regulated by glucose-6 phosphate. Glucose-6 phosphate negatively regulates hexokinase. And that's because if the glucose-6 phosphate levels in the cell go up such that it can't be shunted to glycogen-- something we'll talk about today-- or be sent through glycolysis, it makes sense not to trap more glucose. Once glucose-6 phosphate enters the rest of the glycolytic pathway, the key step of regulation is phosphofructokinase. It, of course, will have positive feedback, saying, let's put carbon into the pathway. If energy charge low, AMP is high, activates it. This molecule fructose 2,6-bisphosphate that we talked about earlier today-- we'll mention again today-- can be involved in some other signaling inputs to allosterically regulate the enzyme. FBP can feed forward to activate pyruvate kinase. You want to match input with output. And, of course, if PEP builds up, you want to slow it down. If a downstream product-- citrate-- builds up, you want to slow it down. And of course, a major output of glycolysis is ATP. And if ATP is high, no reason to continue oxidizing glucose carbons. Now this regulation we discussed is often done at the allosteric level by controlling enzyme rates. That can be via a small molecule binding, as I've shown up here. And it could also be via signaling. And that's really a place where fructose 2,6-bisphosphate as a combination of signaling, and allosteric small molecules comes into play. That is, you can have signaling that controls the enzymes that generate fructose 2,6-bisphosphate, thereby creating an additional molecule that can act as allosteric regulator of the enzyme PFK to generate FBP and commit carbon to glycolysis.

~02:44Now we closed the discussion last time beginning to discuss how it is that organisms can make glucose. Obviously, this glucose carbon that is being oxidized to get ATP has to come from somewhere. We mentioned that plants make glucose during the day when they have plenty of excess energy from light from the sun but then use that glucose to do constant metabolism to maintain their ATP levels at night. And we as animals obviously take advantage of this by eating the glucose that ultimately was created by plants using the energy of the sun. We began to discuss an example of where us, as organisms, also make glucose. Of course, this is not net production of glucose. But for instance, if our muscle cells are working hard, lactate can build up. That lactate is excreted into the blood. That lactate can then be used by the liver to regenerate glucose. And one of the major jobs of the liver is to maintain a constant level of blood glucose to continue to supply fuel for various organs, including the muscle to be able to carry out the work that it needs to do. And so this whole process is called the Cori cycle. And a major job of the liver is thus generating glucose to maintain blood glucose levels. And the pathways used to do this is a pathway that we began to talk about last time called gluconeogenesis. And it's really a discussion of gluconeogenesis. That is, how can one build a pathway in plants, in the liver, wherever, as a way to generate glucose from something like lactate? Now, of course, we know from last time, lactate dehydrogenase can, obviously, it's a redox reaction to interconvert with pyruvate. And what gluconeogenesis really is it's a pathway that cells use to convert pyruvate or really anything else that can come into glycolysis-- and example being lactate. This, of course, can happen by the lactate dehydrogenase reaction. And then once it has that pyruvate, turn that pyruvate into glucose. That's really what we're talking about when we're discussing gluconeogenesis. Now how can we turn pyruvate into glucose? Well, we just discussed the whole pathway where we could turn glucose into pyruvate-- glycolysis. And so this is really the reverse of glycolysis. I said many times that all enzymes are reversible. So why can't we just run the pathway in the opposite direction? Well, hopefully, if you've been paying attention for the last several lectures, you realize there's two problems with doing that. The first is that, remember, why we're doing metabolism in the first place is to maintain a high ATP:ADP ratio under conditions so that the cell can use that ATP:ADP ratio to carry out otherwise unfavorable processes. And so thus, at a very high level, cells can't stop catabolism. Glycolysis or whatever has to continue to maintain ATP in the right range. And so now, if we're going to do something anabolic-- that is, build something, turn it into glucose, this has to happen in the background where catabolism has still happened. And so at least you need some kind of way to control both and prevent this from being some kind of futile cycle. Second, even if that ATP level is super high in the cell, it has extra energy, there is still a thermodynamic problem with just reversing glycolysis. And that is, remember, delta G has to be less than 0 for any pathway to work. And so it then stands to reason that if glucose oxidation to pyruvate, if this reaction is favored, as it is in glycolysis, that delta G is less than 0.

~06:03Well, under the conditions where that's occurring, if we want to then say, well, what about the reverse? Let's switch the signs-- pyruvate to glucose. We know that delta G is the same as it was in the opposite direction but, now, changing the signs from positive to negative or negative positive. And so, thus, delta G, for the reverse reaction, must be greater than 0 and, therefore, must be spontaneous-- is not spontaneous. And therefore says that we need to have a different pathway simply for energetic reasons if we're going to go in the opposite direction. Now oxidizing glucose releases energy. That's how we keep the ATP:ADP ratio high. It stands to reason then that we are going to have to put energy in if we're going to run in the opposite direction. And so if we're going to build a pathway from pyruvate to glucose, you can guess, we're going to have to add couple that pathway to ATP-ADP hydrolysis-- that is, couple it to something favorable to now allow this otherwise unfavorable reaction to happen. Now this energy, of course, in plants can come from the sun. We'll discuss that in great detail when we get to photosynthesis. However, for the sake of you and I, I guess, what it says is, if you think about this, if you release energy from glycolysis, it makes sense then you have to add energy if you're going to do gluconeogenesis. But remember, to satisfy all the laws of thermodynamics, the net energy loss to the universe is going to be-- must exist, which means that it is going to cost more energy to make glucose than you're going to get back from breaking it down. That just, hopefully, fits with your intuition. And it actually, I guess, is good news, because when are we going to do anabolism? When are we going to store all these excess calories as glucose? Well, we're going to do it if we overeat. And if we overeat, and it costs us energy in the form of calories to actually store that energy for later, I guess, it means that when we overeat, we don't have things quite as bad as it could have been. All right. Now let's get back to the how one can actually do this. So if we want to think, what is a pathway that we can build to get from pyruvate back to glucose? Well, we know we're going to have to burn some ATP to do it. But what are the issues with simply reversing glycolysis? Well, we can come back here and say, well, what was driving glycolysis in the first place? And it was these big steps of free energy change acts of hexokinase, phosphofructokinase, and pyruvate kinase. Where as many of these other reactions, at least under these standard conditions that are estimated to exist in cells, are relatively close to equilibrium. So there's really no problem with reversing glycolysis across here or across there. The real issue comes from getting around the hexokinase to phosphofructokinase and the pyruvate kinase step. These are the places where the most energy is released, if you will, when you're going through glycolysis. So those are the steps we're going to have to add energy in order to reverse build a pathway that goes in the opposite direction and allows you to start with pyruvate and end up with glucose. And so what we will find is that gluconeogenesis and glycolysis use many of the same enzymes. However, not all of the enzymes are the same because we need different enzymes if we're going to get around this hexokinase, phosphofructokinase, and pyruvate kinase steps of the pathway. And so for gluconeogenesis, we essentially need four new enzymes in order to get past those places. So we're going to need a new enzyme called glucose 6phosphatase. And glucose 6-phosphatase is going to allow us to get from glucose 6-phosphate and turn it back into glucose.

~09:41All right. We're going to need an enzyme fructose 1,6-bisphosphatase, which is going to allow us to take FBP and turn it back into fructose 6-phosphate. And then we're going to need two enzymes to get around the pyruvate kinase step. These are two enzymes called PEPCK and PC. That stands for phosphoenolpyruvate carboxykinase and pyruvate carboxylase. I'll discuss these in great detail in a minute. And these are going to allow us to get from pyruvate back to PEP. Also, essentially, I'll add them up here to this diagram. So to get back from pyruvate to PEP, two enzymes-pyruvate carboxylase and phosphoenolpyruvate carboxykinase to get from fructose 1,6-bisphosphate, fructose 6phosphate, an enzyme called FBPase. And to get from glucose 6-phosphate back to glucose, an enzyme called glucose 6-phosphatase. OK. Now, of course, if you think back, we're also going to need to balance electrons in the pathway. Remember, glucose to pyruvate is an oxidation reaction. That is, we're generating NADH. That means to go in the reverse direction, we need a source of NADH to reverse that GAPDH reaction. I just want to point out that you're only doing gluconeogenesis if cells have plenty of energy. What does plenty of energy mean? Well, that means the ATP:ADP ratio is high because you're doing lots of metabolism. But if you're doing lots of metabolism, and the ATP:ADP ratio's high, that also means the NADH/NAD+ ratio is high. And so you can think of the NAD regeneration by gluconeogenesis as being, for all intents and purposes, an alternative to fermentation, if you will, as a way to keep catabolism going. That is, you're only doing this if you have plenty of NADH around. And so dealing with that electron balance is less of an issue. OK. Now let's discuss briefly how some of these enzymes work. So glucose 6-phosphate and fructose 1,6bisphosphatase are very straightforward. Effectively, all they are is simply removing a phosphate. That's very favorable. Remember, when we did those reactions of hexokinase phosphofructokinase reactions and glycolysis, that, we had to couple otherwise unfavorable things-- adding a phosphate group to ATP hydrolysis. This is the reverse of that. And so the reverse of that, delta G is going to be less than 0. Delta G less than 0 means we just have a phosphatase there, just like you might have on a kinase enzyme. That's very straightforward. But there's a much bigger issue to get around this other step, this pyruvate kinase step. Remember, equilibrium strongly favored PEP to pyruvate conversion. That was one of the reasons why we could use that step to maintain a high ATP:ADP ratio in cells. Well, now, the issue becomes, how can we trap that phosphate back onto pyruvate while generating phosphoenolpyruvate? How one does that is an issue. Even if the ATP:ADP ratio is high, it's never high enough to reverse pyruvate kinase on its own. And so we need a new pathway with two steps in it. That's why there's two enzymes-- PC and PEPCK-- that are there to create a pathway where pyruvate to PEP now becomes favorable. That is, where delta G 0 prime is less than 0. That is, the equilibrium favors to the right. And under cellular conditions, this pathway can actually happen such that cells can net make glucose. Now these are two steps, both of which require ATP. And so you effectively get one ATP from PEP to pyruvate conversion. It costs you to ADPs or two ADP equivalents to go back from pyruvate to PEP, which makes sense. You should have to spend more energy to go upstream than you get back coming downstream. All right. Here's how this pathway works.

~13:03So this is pyruvate, the first reaction catalyzed by PC, which stands for pyruvate carboxylase. Couples ATP hydrolysis to addition of a CO2 that generates this intermediate, which is called oxaloacetate, often abbreviated OAA for Oxaloacetic Acid. That oxaloacetate is then decarboxylated. That is, the CO2 that is lost is removed-- that was added is removed. This reaction is coupled to GTP and is catalyzed by this enzyme, PEPCK PEP carboxykinase and uses that phosphate from GTP to trap pyruvate in the enol form as PEP or phosphoenolpyruvate. great. So once you've regenerated PEP, now that PEP gets to this flat part, if we go over here to our delta G place. So now it can easily run back across this flat part of the curve. That is, go through enolase phosphoglycerate mutase, phosphoglycerate kinase, GAPDH, taking advantage of the high NADH/NAD+ ratio that would exist when you're doing gluconeogenesis-- triosephosphate isomerase, aldolase. This generates a DHAP and a glyceraldehyde 3-phosphate that you can then combine to give a fructose 1,6bisphosphate. Now we get back here to now we have this steep part there. But rather than going through FBP, we can just have a phosphatase, fructose 1,6-bisphosphatase that releases the phosphate. Now, we have fructose 6phosphate. That fructose 6-phosphate can then go through glucose 6-phosphate isomerase and generate glucose 6phosphate. That now gets back to this other steep hill. So this is relatively flat. Now, you have this steep hill around hexokinase. But rather than coupling it to adding to ATP-ADP hydrolysis, you just remove the phosphate with glucose 6-phosphatase. And in the end, now we have a pathway using these four extra enzymes that give us a way to start at pyruvate and run the opposite direction and generate glucose. And you can do so in a way that is thermodynamically favorable. In other words, we can start with 2 pyruvate and use 4 ATP-- so and ATP to turn pyruvate twice into oxaloacetate. So that's two. And then another ATP at phosphoglycerate kinase to go backwards there to go from-- to run that reaction backwards, plus 2 GTP. Those 2 GTP are effectively the equivalent of an ATP. So this would be 6 ATP equivalents. Why are the GTPs equivalent of ATP? I just want to point out that cells have enzymes that basically can interconvert ATP and GDP or, really, any nucleoside triphosphate and diphosphate to GTP plus ADP. This has a delta G 0 prime that is effectively 0. And so cells basically match their ATP to ADP ratios with any other ratios of nucleoside triphosphates and diphosphates because of this reaction. And so that's why the two GTP are roughly an ATP equivalent. So we'll just call it 6 ATP plus 2 NADH to reverse the GAPDH step. That gives us 6 ADP plus 2 NAD+ plus 6 inorganic phosphate plus a glucose. The delta G 0 prime for this entire coupled pathway is on the order of negative 9 kcals per mol, with the delta G less than 0, equilibrium lies to the right. Net synthesis of glucose is favorable. And so if we just think about this in ATP terms, remember, when we discussed glycolysis, you harvest net two ATP from conversion of glucose into two pyruvate. If we reverse the reaction doing gluconeogenesis, in ATP terms, we have to invest six ATP to turn that pyruvate back into glucose. Makes sense. We didn't make a perpetual motion machine. Both pathways are favorable. But it costs more energy to make a glucose than we can get out by burning the glucose. I should say, the energy isn't just the ATP. There's also this NADH.

~16:23That NADH that's produced from glycolysis also is storing energy, if you will, from oxidation and an NADH/NAD+ ratio, and you're consuming that going in the opposite direction. You only do gluconeogenesis if you have a high ATP:ADP ratio, of course, and a NADH/NAH+ plus ratio-- that is, if you have it under the right conditions to actually run this pathway and have it make sense for the cell. All right. Now I want to discuss, briefly, how these enzymes in gluconeogenesis work. I'm not going to spend time on fructose 1,6-bisphosphatase or glucose 6-phosphatase. As I said before, this is just hydrolysis of that phosphate-alcohol bond. That's relatively straightforward. But I do want to spend time on pyruvate carboxylase and PEP carboxykinase because this introduces a new reaction, carboxylation. But it also introduces a new concept in metabolism that is actually quite important in eukaryotic cells. And that is this issue of so-called compartmentalized metabolism. And, of course, you've learned in introductory biology that a big difference between eukaryotes and prokaryotes is eukaryotes have all of these organelles-- that is, these membrane-bound structures within cells that carry out various functions. So one reason that it's useful to have these different membrane-bound organelles is it creates different compartments within the cell. What does that mean? Well, if you have different compartments, that means you can create different conditions within each compartment. And that's very important for metabolism. Well, why is that important for metabolism? Well, because, remember, whether or not reactions are favorable depend on delta G, which, of course, is dependent on the equilibrium constant, which is related to delta G 0 prime but also to the ratio of the products and reactants within that compartment. And so by having different compartments, you can have different ratios of metabolites-- say, a different ratio of ATP to ATP or a different ratio of NADH to NAD+. Having those different ratios means that because delta G is proportional to the ratio of the reactants over the products-- I'm sorry-- products over reactants, by having different ratios of things like ATP and ADP, NAD, NADH, you can make different reactions more or less favorable depending on which compartment in the cell that they're located. Now it turns out that glycolysis and most of gluconeogenesis takes place in the cytosol of eukaryotic cells. The cytosol is basically the space inside the cell that's not inside another organelle. However, pyruvate carboxylase is a reaction that takes place in mitochondria because mitochondria have a particularly high ATP:ADP ratio. And it turns out, this helps favor the pyruvate carboxylase reaction. And so we'll talk a lot about reactions in the site cytosol versus the mitochondria. But basically, here's glucose to PEP. Whether you do that in one direction by glycolysis or the other direction by gluconeogenesis, that happens in the cytosol as does turning that PEP into pyruvate. But if you're going to reverse the reaction, eukaryotes take advantage of a different compartment, the mitochondria, with a high ATP to ADP ratio. And basically, in the mitochondria, turn that pyruvate into oxaloacetic acid-- that is, carry out the pyruvate carboxylase reaction. And then that pyruvate carboxylase reaction generates oxaloacetic acid, and then PEPCK can, either in the mitochondria or in the cytosol, generate PEP. And so here's a place where one can run one reaction-- PEP to pyruvate of t make ATP in the cytosol-- but a different reaction-- pyruvate carboxylase to turn pyruvate into oxaloacetic acid that would then be used for PEPCK to turn that back into PEP and have that take place in the mitochondria.

~19:39I want to point out, just so for MCAT exams and things like that, PEPCK is classically defined as a cytosolic activity, at least in terms of gluconeogenesis. Although, there is a mitochondrial isoform of PEPCK. And it's at least somewhat debated. There's some evidence that, at least, in some tissues, that might be important for gluconeogenesis as well. And so that's why I draw it as PEPCK using oxaloacetate to PEP either in the mitochondria or in the cytosol because that's an area of active investigation right now. All right. Let's start by talking about how does pyruvate carboxylase work? This is an example of a carboxylase reaction, that is, adding a CO2 group to a molecule to make a carbon-carbon bond. That purple CO2 is added to the CH3 of pyruvate of to make oxaloacetate. And how this happens, in many cases, uses a cofactor. And that cofactor is called biotin. Biotin, like many cofactors, is a vitamin. And this provides a useful functional group. In this case, enzymes that use biotin use ATP to basically drive CO2 addition to biotin. And then that CO2 on the biotin is then activated to be transferred to another molecule in a carboxylation reaction. All right. So what does biotin look like? I will draw it for you. So this, here, would be biotin. I've drawn it in the enolate form of biotin. I'll draw it in a different way in a second. Biotin is typically bound to a lysine molecule in the active site of an enzyme that uses this. And so this, here, is basically a peptide bond between the terminal amino group, the epsilon amino group of the side chain of a lysine in a residue that basically makes a peptide bond to link biotin into the active site of the enzyme that's using it. Biotin is often drawn in the keto form. And the active part of biotin is basically this top part of the molecule. And so to draw it to see differences between the enolate and keto form, I just want to show that quickly. I drew it in the enolate form because it's easier to see, I think, how it-- so this, here, would be the keto form where biotin is drawn most commonly. And basically, the reaction, the way biotin picks up a CO2 is as follows. And that is CO2 can exist particularly under basic conditions as CO2 or as-- this is bicarbonate. OK. So bicarbonate-- turns out, some enzymes use CO2 directly. Some enzymes use bicarbonate. It turns out that pyruvate carboxylase uses bicarbonate. So biotin bound in the active site of pyruvate carboxylase will utilize bicarbonate in the following way. And I'll just point out that the pH of the mitochondria is also more basic, which also helps this pyruvate carboxylation reaction. All right. So what happens is this bicarbonate is phosphorylated by ATP. This phospho intermediate can then react with this active site here of biotin to release the phosphate. And now, you now have this activated CO2 group that's attached to the biotin. Here's pyruvate drawn in the enol form. You can look back in your notes about how to interconvert it between keto and the enol form. I've shown that several times. And then this, then, can end up adding this CO2 to the end of pyruvate to generate oxaloacetic acid plus regenerate the biotin cofactor in the right form. It'll be the keto form, which can then get back to the enol form. And it's ready to do another catalytic cycle. And so, basically, pyruvate carboxylase uses ATP to produce this carboxylate of biotin. And then uses that to add the CO2 to pyruvate to generate oxaloacetic acid. Now I want to point out that oxaloacetic acid is both an alpha keto acid like pyruvate was. And it's a beta keto acid. And so this ketone is alpha to that carboxylic acid. And it's beta to that carboxylic acid. So it's both an alpha and a beta keto acid. And it turns out that the way PEPCK works is it takes advantage of the favorable decarbonization of a beta keto acid, which we talked about last time, along with GTP to phosphorylate pyruvate and trap it in the enol form.

~23:33And so this is how PEPCK works. So I'm going to draw GTP in this very stylized way-- so guanine with three phosphate groups. There's oxaloacetate. That decarboxylates-- takes advantage of this being a beta keto acid to decarboxylate and add the phosphate. And that's how we can generate PEP. OK. Great. And so that's how you can build a different pathway that uses ATP and GTP for energy input in order to run glycolysis in the opposite direction-- gluconeogenesis. All right. So how is gluconeogenesis regulated? Well, just like the principles we talked about in glycolysis, it works in a way that makes sense. And so you can guess that the steps that are going to be regulated are exactly the ones that you might guess. They were the same steps that were regulated running glycolysis. It's going to be these big changes where energy changes occur across the pathway, which are also the entrance and exit of the pathway. And basically, regulation has to be reciprocal. So in glycolysis, you want to increase glycolysis under conditions where you need to produce ATP. And you want to decrease glycolysis if you have enough ATP or enough of another downstream product, such as citrate. All right. Well, gluconeogenesis is going to be exactly the opposite. You certainly don't want to do gluconeogenesis if you need energy release. If you need ATP, you don't want to run gluconeogenesis. However, you want to increase gluconeogenesis, if the cell has ATP excess, you also want to do it if you have excess of other products like citrate because why go through the trouble of sending things down glycolysis if you have nowhere to put it. You might as well, instead, make glucose or shunt that glucose off to produce glycogen. And so let me just add that regulation here. And so major regulators of gluconeogenesis are as follows. And so high levels of citrate is going to stimulate fructose 1,6-bisphosphatase. So high levels of citrate will inhibit things coming through glycolysis and activate gluconeogenesis by acting at fructose 1,6-bisphosphatase to match the energy considerations. If you have high levels of AMP, energy charge is low. You want to stimulate glycolysis at phosphofructokinase. Similarly, that's going to inhibit gluconeogenesis at FBPase. And it turns out, high levels of ADP-- I'm sorry-- yeah, high levels of ADP, low energy charge, also inhibits PEPCK because you also don't want to try to generate PEP under those conditions. Great. All right. Now in animals, we also want to regulate glucose catabolism and production under signaling control. And this gets back to this other topic we talked about, such as the liver's job being to maintain a constant level of blood sugars. Most of you are very familiar with blood sugar control through diabetes. You've probably heard that this is under hormonal control, that the job of insulin is to lower blood glucose. And you also have hormones like epinephrine or glucagon whose job is to raise blood glucose. Why do you want to raise blood glucose? Well, if you have some kind of fight-or-flight response-- you see that lion, and you need to run away, your body has this adrenaline rush. That's what epinephrine is. That basically wants to supply more energy to your muscles, so that you can run away effectively. So it turns out, both insulin and glucose act on lots of tissues in the body. I'm going to focus on what they do in the liver. And that's because in the liver, this is a major organ that regulates blood glucose levels. So if you have excess glucose around, what do you want to do? You want to stimulate glucose uptake into cells, its metabolism, and its storage. And so insulin with high blood sugar basically wants to stimulate, taking glucose into the liver and storing it as glycogen.

~27:03If you want to run away from the lion, well, now you have epinephrine around. You want to make sure that as you're consuming that glucose in your blood that you're continually making more. And so you want the epinephrine to stimulate release of glucose either from gluconeogenesis or from the breakdown of glycogen. And both of these hormones act-- insulin and epinephrine-- act at a level where they can regulate enzymes of glycolysis and gluconeogenesis as well as entry and exit of glucose monomers in and out of glycogen. And so this is an example of how fructose 2,6-bisphosphate works. And so remember you learned from Professor Yaffe that epinephrine acts via cyclic AMP signaling, which turns on a kinase-- protein kinase A. And that protein kinase A can regulate enzymes that produce or break down, basically, these enzymes that produce or break down fructose 2,6-bisphosphatase which, in turn, regulates PFK activities such that you can match your need to burn that glucose versus doing gluconeogenesis in the case of the liver, so you can do enough gluconeogenesis in order to have glucose around for the body to use in that fight-or-flight response being driven by epinephrine. Now a major effective insulin and epinephrine signaling, though, is actually on release or storage of glucose molecules in glycogen. And I alluded last time in talking about the regulation hexokinase that glucose 6phosphate is basically the entry point to get glucose units in and out of glycogen. And I want to discuss how you add and subtract those glucose units into storage polymers. Now I'm going to discuss it in the context of glycogen storage in humans. But remember, plants actually store things also as glucose polymers. The chemistry is very similar. But, obviously, the regulation is very different.

~28:41Now you'll hopefully recall from past lectures that these storage polymers of glucose-- glycogen in humans, starch in plants-- are basically these alpha 1,4 linkages of glucose molecules. Remember, there was a nonreducing end and a reducing end of the molecule. So starch was a straight-chain polymer. Glycogen had these alpha 1,6 branch points that made this branch polymer, where there's lots of non-reducing ends and a single reducing end. And each of these non-reducing ends is a polymer of glucose molecules with this alpha 1,4 linkage that, at the branch point, has this alpha 1,6 linkage to make this long-chain branched polymer. Now when we discussed this at the time, I mentioned that this is useful because you have all these non-reducing ends that can be used to add or subtract glucose monomers. And that's great. This is a nice compact form of storage with lots of places to put or remove glucose from. You either store it quickly, insulin driving glucose storage-- or remove it quickly, epinephrine driving release of glucose from the glycogen. And so if we're going to have a way to add glucose polymers or break them down, those are two separate pathways, just like glycolysis and gluconeogenesis-- reciprocal activities, but we need two pathways to do it because delta G has to be less than 0 for each pathway to work. Also, we have to be able to regulate these separately because we don't want to have a futile cycle. Now the way you add and subtract glucose polymers to glycogen acts through glucose 6-phosphate being first converted to glucose 1-phosphate. So this, here, is alpha glucose 6-phosphate. Remember, it's alpha because I drew that hydroxyl at the one position pointing down. This, obviously, can enter glycolysis and come in from glucose via hexokinase. All right.

~30:19This is first converted via a mutase reaction to move the phosphate from the 6 to the 1 position of glucose. So this, here, is glucose 1-phosphate. The way the mutase reaction works is exactly the same analogous mechanism that I explained for phosphoglycerate mutase in glycolysis-- moves the phosphate in this case from the 6 to the 1 position of glucose. And then once you have that glucose 1-phosphate, that can be added to a non-reducing end of a glycogen polymer using an enzyme called Glycogen Synthase, GS. So that'll give me a glycogen molecule with one additional monomer added to the non-reducing end of the polymer. And you release that glucose back off via a different pathway, a different enzyme called phosphorylase. All right. Now the activity of these enzymes-- glycogen synthase and phosphorylase-- that is, the pathway to add glucose 1-phosphate glucose monomer to the polymer versus remove it from the polymer is under hormonal signaling control in animals. And basically, that hormonal signaling works as follows. So phosphorylase can be phosphorylated or dephosphorylated via a signaling enzyme, an enzyme like phosphorylase kinase, which is downstream of PKA, which is downstream of epinephrine signaling. OK. And so when phosphorylase is phosphorylated by phosphorylase kinase-- that has, has a phosphate group added is a signaling cascade-- it is in the active state. And when it's dephosphorylated, it's in the inactive state. Well, glycogen synthase also is subject to regulation by protein phosphorylation on the enzyme by a kinase. Except this has the opposite relationship. So when it is in the phosphorylated state, it is inactive. But it is in the non-phosphorylated state, it is active. And so protein kinase A, which is downstream of epinephrine, can both activate phosphorylation of phosphorylase and glycogen synthase. And that makes sense. You want to release glycogen glucose monomers from glycogen. You turn on, by phosphorylation, the pathway to release them. And you turn off the pathway to store them. Another kinase that phosphorylates to glycogen synthase is a kinase called GSK or Glycogen Synthase Kinase. This kinase is inhibited by insulin signaling. And so this is a negative of a negative. So effectively, a negative of a negative will keep it in the inactive state. And so insulin, by inhibiting the ability of GSK to put glycogen synthase in the inactive state, will make glycogen synthase active. And when you have high insulin around, that actives glycogen synthesis, and you store glucose monomers as glycogen. Get them out of the blood. OK. Now let's discuss the chemistry that allows us to do this glycogen synthase and phosphorylates reactions. Now as we go through this, what you will see is that when we break down the polymer, energy is released. So that's going to be breaking down a polymer. That's the right direction of entropy. And so that's going to be released. And storing it is going to require energy. That is, you're going to have to have some energy input to build a polymer. But nature actually does this in a way that is actually quite efficient. First, let's talk about how you store glucose 1-phosphate units by adding them to glycogen. So this uses energy input from UTP, which is an ATP equivalent for exactly the reason I described earlier. That is, because ATP plus UDP, like any other interconversion of nucleoside triphosphates and diphosphates is very close to equilibrium.

~33:26So the UTP to UDP ratio should be similar to the ATP the ADP ratio. And so UTP is really an ATP equivalent. But nature, for whatever reason, decided to use UTP here. And it generates a nucleoside sugar called UDP glucose. And so let me show you what that is. So here is glucose 1-phosphate. I will draw a stylized version of UTP here. And so this molecule with the UDP added to the glucose is UDP glucose. This will also generate a pyrophosphate. And that pyrophosphate can be hydrolyzed to generate two inorganic phosphates, effectively, like building any other polymer, by doing this downstream-- keeping that product of the first reaction low helps pull that reaction forward for polymer synthesis. This UDP glucose can now react with the non-reducing end. So this, here, will be the non-reducing end of the polymer. That's the OH group at the 4 position, at the nonproducing end. And so that then generates that alpha 1,4 bond to add to the polymer. So here, we have glycogen n plus 1 plus releases UDP. And so the net to add a glucose 1-phosphate to the non-reducing end of the growing glycogen polymer is net conversion of a UTP to a UDP. So that is one ATP equivalent to add glucose 1-phosphate to the end of the polymer. Now, of course, you had it to also have an ATP from hexokinase to capture that glucose 6-phosphate to begin with. But effectively, this is two ADP molecules to add a glucose to glycogen. All right. Now if we're generating starch, that's all we have to do, just build a long polymer. But if we're making glycogen, remember, glycogen has all of these branch points on them as well. And to do the branch points, you basically have to make this alpha 1,6 bond, which then gives you two new non-reducing ends that you can add more subunits to. The way nature does this is as follows. And so if I just draw this here in a stylized way, it turns out, once you get to about seven units here in the growing polymer-- so this is the reducing end, and this is the non-reducing end of the polymer. And so the polymer is growing by adding things to that end. Once you get about seven, there's an enzyme that will basically cleave this, and move it and make a new alpha 1,6 bond. OK. Here we go. We have our reducing and our non-reducing end and basically transfers these 7 units over. And now, we have two non-reducing ends that we can continue to grow the polymer from. You can think of this like the airplane analogy. When you load an airplane, at least the way that's most efficient, the people who are sitting in the back of the plane, they get on first. But then, they're the last people off the plane because you add basically to these non-reducing ends, and then you subtract from those non-reducing ends. And so the first glucose added, the one at the reducing end, is the last one that is going to be removed. All right. Now phosphorylase is the enzyme that allows you to do the opposite-- that is, to break that alpha 1,4 bond and release monomers. And it's called phosphorylase because it uses phosphate to break that alpha 1,4 bond, taking advantage of the fact that breaking down the polymer is favorable and actually releasing the glucose from it again is a glucose 1-phosphate. And so at the non-reducing end of the molecule-- so this, here, would be the non-reducing end of the glycogen polymer. You basically have a phosphate molecule that takes that off, so you have a glycogen n minus 1 plus a glucose 1 phosphate, which can now undergo a mutase reaction back to glucose 6-phosphate that could then be released from the cell if it's a liver, or it could be burned in glycolysis.

~37:02Now, obviously, if you're doing breakdown, that is, your chewing back from non-reducing ends, eventually, you're going to hit a situation where you come to where there's just a nub here with an alpha 1,6 bond there. That is, chew it back till you hit this single monomer with a non-reducing end here to this alpha 1,6 break point. Turns out, you just cleave off that nub, if you will. And so that nub is then just released as free glucose. So other than release of that nub, what it means is that you actually basically get no ATP to actually break down in the phosphorylase reaction. So the net cost to store a glucose 1-phosphate molecule is one ATP via the UTP to UDP conversion in the glycogen synthase reaction. And you get that one glucose 1-phosphate back. And so two ATPs to put a glucose tin glycogen. And then you get it directly back out as a glucose 1-phosphate. That's a phosphorylated glucose that can then enter glycolysis. So that's one less phosphate you need to spend on glycolysis. And so it's incredibly efficient to store glucose in that way. And so it's actually slightly greater than one ATP to store it as glycogen because you lose in ATP with that nub removal at the branch polymer. But for all intents and purposes, it costs one ATP to store your glucose as glycogen, which is pretty amazing. All right. Now before I leave the topic of sugar storage, I want to make some comments on some sugars that are stored in forms other than glucose. And want to talk about these because these sugars are, of course, important parts of our diet. And so as we've mentioned in prior lectures, two of these sugars are sucrose and lactose. So remember, sucrose is a disaccharide of glucose plus fructose. And lactose is a disaccharide of glucose plus galactose. So those are storage forms of sugar-- sucrose in the case of plants, lactose in the case of milk made by mammals. And so to break those sugars down, basically, you break the disaccharide. Now, you have the glucose molecules you know how to handle. That's glycolysis. But I want to spend a little bit of time how you deal with the fructose and the galactose that you get out from these sugars because this actually relates to some things that I'm sure you've all read about in the news. For instance, fructose as a sugar in our diet is actually quite controversial. Lots of stuff out there that says fructose was cooked up in the devil's kitchen and is this evil thing. There's actually others who argue that, oh, fructose is the same as glucose, the same number of calories. There's both sides of the debate. Both sides are actually making factual claims. And I want you to understand the biochemistry, so you can actually judge for yourself who's right in those claims. All right. So let's start with talking about how you metabolize fructose. And so fructose is first captured by phosphorylation to generate fructose 1-phosphate. This is carried out by an enzyme called ketohexokinase. Ketohexokinase is an enzyme this present in your gut and your liver. And so fructose in the diet, from breaking down sucrose or from high-fructose corn syrup, whatever, is captured with ketohexokinase to make fructose 1-phosphate. Turns out that phosphofructokinase, our old friend from glycolysis, is less efficient than ketohexokinase but can also carry out this reaction. And remember, what is it do in glycolysis? It adds a phosphate to the 1 position of fructose 6phosphate. Well, it can also add a phosphate to the 1 position of fructose give you fructose 1-phosphate.

~40:24OK. Once you have this fructose 1-phosphate, which, remember, is not in glycolysis, it can be a substrate for the glycolytic enzyme aldolase. What does aldolase do in glycolysis? It splits FBP into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Well, here, you only have a phosphate on the 1 position. And so if you look back at how aldolase works, what you'll see is that if aldolase acts on fructose 1-phosphate, you'll generate a dihydroxyacetone phosphate. That phosphate will go to the dihydroxyacetone half, which, of course, is perfectly good substrate for glycolysis. The other product of aldolase acting on fructose 1-phosphate is just glyceraldehyde without the phosphate on it. And then there's another enzyme that will phosphorylate glyceraldehyde to generate glyceraldehyde 3phosphate, which can then also, of course, enter glycolysis. And so fructose is a hexose just like glucose. You generate two trioses-- DHAP and glyceraldehyde 3-phosphate. That costs you two ATP to do it, exactly the same cost is what you spent to get glucose to make DHAP and glyceraldehyde 3-phosphate. And so people are absolutely right when they say metabolizing fructose has exactly the same caloric cost as metabolizing glucose-- two ATPs to get it into the trioses. And then whatever you get out of it, you get out of it two ATPs, four ATPs-- so net two ATPs if you just do fermentation, more if you do complete oxidation. But the bottom line is that from that standpoint, you get exactly the same number of calories from burning glucose versus fructose. However, I want to point out that the regulation will not be the same. And that's because in this fructose metabolism, you will notice that there was no glucose 6-phosphate and no FBP generated. That means you are avoiding all of the feedbacks that control how much glucose you send to glycolysis-- first to glycogen. Remember, that's what glucose 6-phosphate does, acting on trapping sugar. And more importantly, you're actually not using phosphofructokinase in a way to fit with the rest of the feedback regulation. I also show that here on this slide. It's basically a better version of what I drew up here. And I now want to point out this storage of citrate and this feedback regulation of citrate. So we'll talk about the specifics of this more in other lectures. But it turns out, citrate is the precursor for how one stores carbon as fat. And so if you're putting fructose glucose into the system and citrate is too high, it'll shut off phosphofructokinase and stop carbon coming down the system, shunting it instead to glycogen or just saying don't give me any more glucose at all. If I'm doing the same thing with fructose, those feedbacks don't exist. Citrate doesn't feed back on any of the enzymes that are regulated for fructose metabolism. And so the idea is carbon keeps getting dumped into citrate, which ultimately ends up as fat. And this really underlies, at least on a theoretical basis, why there may be a connection between fructose and obesity or metabolic syndrome. I've posted articles on Stellar, one from The Popular Press, one from a scientific journal that discusses the controversy, discusses this. But now, at least, you have the background to draw some of your own conclusions. OK. That's fructose metabolism. What about galactose metabolism? Well, galactose metabolism interfaces with, effectively, glycogen metabolism or, at least, glucose 1-phosphate-or sorry, UDP glucose and does so in a way that is a little bit non-intuitive. But it's actually very important to understand galactose metabolism, at least for those of you who are looking to go to medical school.

~43:42It turns out that galactose metabolism and issues with metabolizing galactose are what underlie a set of rare genetic diseases leading to a set of conditions called galactosemias. I guess, if you're a pediatrician, you may encounter these. If you're not, these are quite rare. Although med schools love to ask about these, and certainly, they're very popular on things like board exams. So it's something you should know about. And so I just want to briefly introduce galactose metabolism to you.

~44:09So this is galactose. Like all sugars, galactose is initially trapped by phosphorylation. It's phosphorylated on the 1 position to trap it, just like fructose. And so this is galactose 1-phosphate. Remember, galactose is an epimer of glucose. So it differs from glucose here by the stereochemistry of the hydroxyl group at this 4 carbon. And so the way galactose, once it's trapped as galactose 1-phosphate is metabolized is it will react with UDP glucose. So here is UDP glucose from glycogen metabolism drawn in a very stylized way. That will generate a UDP galactose and a glucose 1-phosphate, which, of course, can enter glycolysis, can be stored as glycogen, et cetera. This UDP galactose can then be turned into UDP glucose via an epimerase reaction. And so remember, the epimerase reaction needs to change the stereochemistry of the hydroxyl group at this 4 position, such that it's pointing up in galactose or pointing down in glucose. The way epimerases work is that they require a cofactor. That cofactor is NAD. and they work in the following way. And so if I just draw, here's the 4 position of galactose, the 4 carbon of galactose, with the hydroxyl group pointing up, an epimerase can basically remove a hydride from the bottom face of the molecule, transfer those two electrons to NAD to generate NADH. Now, you get this lactone intermediate at the 4 carbon. And then you can have two electrons from an NADH can be added to the opposite face of the molecule that regenerates an NAD+. And now, we've effectively changed the stereochemistry at the 4 carbon to now be pointing such the hydroxyl points down, and that's glucose. And so the way epimerases work is they basically remove and add electrons from different faces of the pyranose ring. And that changes the stereochemistry of which direction the hydroxyl group is pointing. And so effectively, this is how we metabolize all kinds of different sugars, either putting them in glycogen or other starch, some other storage carbohydrate that we can get them later, and then use them in glycolysis as a way to drive glucose oxidation as a way to release energy and keep ATP high in the cell. All right. Now I want to shift topics and begin to discuss how we can oxidize pyruvate from glycolysis to make CO2 as an alternative to how we can release energy from carbohydrate oxidation rather than just use fermentation. Now you'll see that the enzymes and ways that we do this is going to also be how we make citrate. And so this isn't just about releasing energy. It's also about storing energy because you'll see, citrate is a precursor for storage as fat. The reactions downstream of pyruvate oxidation also make lots of other useful intermediates that can be used to synthesize other things cells need-- amino acids, nucleic acids. And so this pathways we're going to discuss are going to come up over and over again throughout the course. Now to facilitate this discussion, I'm really going to at least initially focus on catabolism.

~46:58And so just to remind you, if we do glycolysis by turning glucose pyruvate, this involves oxidation. So we need to dispose of those electrons somewhere. In other words, that NADH has to be regenerated NAD. Fermentation of pyruvate into something like lactate is a way to do that and allows net ATP production from glycolysis without the need for any oxygen. However, if we're going to completely oxidize that pyruvate CO2 we're going to generate many more electrons as waste that we have to put somewhere. Oxygen is a particularly good electron acceptor. And so we can reduce oxygen to water as a way to deal with that electron waste, and that allows us to, just burning wood, release lots of energy from glucose by transferring those electrons ultimately to oxygen. And of course, that same process-oxygen to water-- if we're using the pyruvate to fully oxidize it, also is necessary to regenerate the NAD to run glycolysis. Now ultimately, the series of reactions that allow chemically conversion of pyruvate to CO2 is called the TCA cycle for try Tricarboxylic Acid Cycle or sometimes referred to as the Krebs cycle and also is referred to as the citric acid cycle-- so TCA, Tricarboxylic Acid Cycle, Krebs cycle, citric acid cycle-- all synonyms for the same thing. What these cycles are is basically a way to take two carbon units. And those two carbon units can come from the oxidation of pyruvate or from the oxidation of lots of other fuels. And those two carbon units are combined with four carbon units. You'll see oxaloacetate is the four-carbon unit-- oxaloacetate gluconeogenesis. That oxaloacetate could come from the pyruvate-carboxylase reaction. Or it could come, as you'll see, from the TCA cycle itself. This generates a six-carbon molecule, which is citrate. Citrate is a tricarboxylic acid, hence the citric acid cycle or the tricarboxylic acid cycle. And then those six carbon units are reoxidized, releasing two CO2 molecules, back to four-carbon oxaloacetate. And hence, this is a cycle. Now this cycle was initially described by Hans Krebs, who predicted the cycle largely based on chemistry-- one of the most amazing feats in the discovery of metabolism. Turned out, he was right. And thus, it's also referred to as his name, the Krebs cycle. Now lots of oxidation is happening in this cycle. You're oxidizing the molecule, releasing CO2. That means lots of energy is released. Those electrons ultimately will be transferred oxygen. And so we can use this to make lots of ATP and do lots of other work. Where are these reactions all occur is in the mitochondria, specifically in the matrix of the mitochondria. Remember, the mitochondria, from cell biology, is a double-membrane organelle. The innermost part of the mitochondria is referred to as the matrix. And these have conditions that favor pyruvate carboxylase reaction, as I talked about, but also favor other oxidation reactions, such as those that occur in the TCA cycle. And so next time, what I will start with is by being more explicit about what chemistry is happening to allow these two carbon units to combine with oxaloacetate 4-carbon to generate citrate and go over the reactions that then allow you to run a cycle to regenerate oxaloacetate, that is, run the TCA cycle as a way to oxidize carbon. Thanks.

🎬 MIT 7.05 Lec 23 · TCA Cycle II, Isotope Tracing & Glyoxylate Cycle⏱ ~50:00

~00:00MITOCW | 23. TCA Cycle II

~00:02MATTHEW

~00:02Hello, everybody. Last time, I introduced the idea of the TCA cycle, tricarboxylic acid cycle. Also known as the

~00:10VANDER

~00:10citric acid cycle, because citric acid is a tricarboxylic acid, as you'll see later today. Also known as the Krebs

~00:18HEIDEN:

~00:18cycle, named after Hans Krebs, who discovered it in the early part of the last century. The TCA cycle is the series of reactions that occurs in the mitochondrial matrix and it allows the complete oxidation of two carbon units, derived from many things, including pyruvate, derived from glucose and glycolysis and enables the complete oxidation of that carbon to CO2. Now, it's a cycle because those two carbon units from pyruvate or other sources enter the cycle, combine with 4carbon oxaloacetate, and combine to make 6-carbon citrate. Hence the citric acid cycle, or TCA cycle, that is then oxidized back to 4 carbon units, forming a cycle that allows cells to release lots of energy. Complete oxygen enables, ultimately, complete oxidation of glucose to CO2. We've talked many times how this releases energy. And it also generates lots of intermediates for the cell that can be used to make other stuff. Last time, I alluded to the fact that citrate can be used, say, to make fatty acids. Today, what I want to do is I want to dive into the details of how this TCA cycle occurs and the consequences of the way it works, and how that affects other aspects of metabolism. You'll see that it actually affects the ability of different organisms, whether or not they can make things from intermediates in this cycle, because of how the cycle works. Now, to start. Of course, if we're going to start from pyruvate. Pyruvate, of course, has three carbons. And so if we're going to generate a two-carbon acetate group, we have to lose CO2. And as I described a couple lectures ago, acetate is also the thing that we can derive from metabolism of ethanol, showing us two things-- metabolized glucose to pyruvate or ethanol itself-- can both be turned into acetate. But really, the donor is this molecule, acetyl-CoA. Which is basically also a carboxylic acid, but rather than being a carboxylic acid, instead makes this a thioester bond, which activates this carbon as a leaving group, such that it can combine with oxaloacetate, which you'll remember from gluconeogenesis, releasing that S-CoA molecule to generate six-carbon citric. So, this is citric. You can see that we've made a bond from this carbon on the acetate, losing this S-CoA group to this carbon oxaloacetate to make this six-carbon citrate molecule, which is a one, two, three-- three-carboxylic acid, or tricarboxylic acid. Hence the name citric acid cycle, tricarboxylic acid cycle.

~02:58Now, these six carbons can then-- or this six-carbon citrate molecule can then be oxidized, generating two CO2 molecules that are released, and ultimately reforming oxaloacetate that can pick up another two-carbon acetylCoA to generate another citrate, and around and around the cycle goes, allowing in the end the net entry of two carbons, effectively from acetate, and release of two carbons as CO2. Now, as I alluded to, this can come from pyruvate. It can come from acetate itself, vinegar. It can come from alcohol. It turns out that when you break down fat, you also break it into two carbon units. And so this cycle becomes very useful for cells, because it allows the oxidation of many different molecules to completely turn that carbon into CO2. Now, if we're going to do this from glucose, however, you'll remember that pyruvate has three carbons. And so if we're going to turn pyruvate into acetate, or acetyl-CoA, we have to lose a carbon of CO2. We have to lose this carbon as CO2. Now, we saw this before, that we can do this via-- this is exactly how we generated ethanol when we did fermentation of pyruvate to ethanol, but that molecule didn't generate acetate, It generated acetaldehyde. The difference, of course, being whether or not this carbon gets oxidized in the case of acetate to the acid, or in acetaldehyde in ethanol metabolism, where it remains an aldehyde. Now obviously, you can make ethanol and then turn that ethanol into acetate. And that's a pathway that I guess certainly would work. However, that's not the way it happens in most organisms. Most organisms actually directly produce acetate, or more correctly, acetyl-CoA from pyruvate. So let's take a look at that reaction. So here once again is pyruvate. And if we turn that pyruvate into this two-carbon acetyl-CoA, let's look here what has to happen. Well, the first thing is that we have to decarboxylate this molecule. And so that generates a CO2. We have to lose that one carbon. Also, as I pointed out, if we do this as if we did it in ethanol metabolism, we'd be left with an aldehyde. But this is an acid, and so this carbon also has to be oxidized. We know how to do that. We can donate those electrons to something like NAD, so the NAD is reduced to NADH. And we had to add this S-CoA molecule, which I'll come to in a minute. And so each of these steps ends up making a fairly complicated reaction. Obviously, several co-factors are going to be involved. You should be able to guess that just by looking at it. Obviously, I already drew up there NAD. If we're going to decarboxylate, remember this is a alpha carboxylic, an alpha ketoacid-- the ketone group is alpha to the carboxylic acid. And so if we're doing alpha decarboxylation, as you might guess, we need a cofactor. That co-factor, as I told you before, is the co-factor factor thiamine pyrophosphate. And finally, there's this S-CoA group. We need to describe what that is. However, before delving into those, and there's actually a couple other factors that are needed as well, I want to mention one other issue about this reaction, in that this reaction happens in the mitochondria, because that's also where the TCA cycle happens. So, recall that if we divide the cell into two compartments like an eukaryotic cell, here we have the cytosol and the mitochondria in a eukaryotic cell. As we've already talked about, having different compartments helps facilitate different metabolic reactions, because you can have different conditions in the two compartments.

~06:54And so as we said, glycolysis occurs in the cytosol. Glucose to pyruvate. And last time I mentioned, the TCA cycle occurs in the mitochondria. And so that means if we're going to fully oxidize the pyruvate carbon to CO2 using the TCA cycle in the mitochondria, that pyruvate has to get from the cytosol to the mitochondria, or at least carbons from the pyruvate have to get there. It turns out, you'll see in a minute, acetyl-CoA is a very large group. And so pyruvate itself is transported into the mitochondria. And that's where the reaction occurs to turn it into acetyl-CoA that can then enter the TCA cycle and oxidize. However, what this means is that a transporter is actually needed to get this across the mitochondrial membranes and into the matrix of the mitochondria where the TCA cycle happens. And I like to mention this because it turns out the pyruvate carrier-- that is, the transporter-- the way that it actually gets that pyruvate from the cytosol into the mitochondria actually was an unknown thing about metabolism until 2012, so not that long ago. Sometimes one can be left with the feeling, listening to these metabolism classes or reading a textbook, that everything about metabolism has been known forever, but it's actually not true. Here's a very key, central part of the pathway that was actually just discovered, at least at the time of this lecture, less than 10 years ago. It also illustrates that not all metabolism is completely understood. Now, let's get back to this reaction, how you turn pyruvate into acetyl-CoA. And I want to go through now and point out that several co-factors are needed. And so I already mentioned one of them, S-CoA, which is shorthand for coenzyme A. So, I need to tell you what that is. You hopefully are already familiar with TPP plus, thiamine pyrophosphate. We talked about that when we talked about how you do alpha decarboxylation to generate ethanol from pyruvate, also used here for the alpha decarboxylation reaction. Redox reaction happens, and so we needed NAD plus to get converted into NADH. We talked a lot about how that serves as an electron carrier, but it turns out that there's two additional electron carriers that are involved in this reaction. One of them is called FAD and the other one is called lipoic acid. Now you might say, why do we need all these electron carriers? Well, these are just different molecules that can carry two electrons, similar to NADH. And effectively, what these can do by having multiple electron carriers-- one can build chains of oxidation and reduction reactions. And it turns out these chains of oxidation reduction reactions really become central to energy transfers in biology, because building these chains allows more easy stepwise release of energy as one moves across these oxidation reduction reactions. Which remember, as I alluded to earlier, really are at the core of bioenergetics and a lot of what allows energy release from these pathways. What I mean by this will be more explicit as we go through what some of these co-factors look like. So, I'm not going to draw TPP plus or NAD again, but let's define what some of these other cofactors look like. So, let's start with coenzyme A. So, coenzyme A, it turns out, is useful. It's actually involved in lots of acylation reactions.

~10:34What's an acylation reaction? Well, that's basically if you're making a carbon-carbon bond by adding a molecule of greater than one carbon, so two carbons or greater, to something else. That's an acylation reaction, as we did with adding the two-carbon acetate to oxaloacetate to make citrate. And you'll actually see coenzyme A will come up in this in many, many lectures throughout the rest of the course.

~11:00Why this becomes useful is because it activates this basically carbon to the left of, in this case, the ketone. Because, by having that thioester there, ends up activating that carbon and allows it to carry out these acylation reactions. Coenzyme A itself is derived from a vitamin called pantothenic acid. And as a vitamin, this is something that you have to get from the diet. Now, when we abbreviate coenzyme A, which is often abbreviated S-CoA, you get the sense that it's this little tiny molecule that's just stuck on sulfur-- stuck on to the end to make this thioester bond. But it turns out coenzyme A is actually a giant molecule, one of the reasons why you actually synthesize acetyl-CoA in the mitochondria, because it's hard to transport this giant molecule. And so this is what pantothenic acid looks like. So, if I put an acid group here and a hydroxyl group there, that would be pantothenic acid, the thing that's in your cereal, on the side of your cereal box. Then there's these additional pieces. This is the active end of the molecule, that's that sulfur from the S-CoA, and then this side of the molecule is esterified to two phosphates, which are esterified to-- I'm not going to draw it out, but this would have an adenine base and a phosphate there. So basically, this is ADP, with a phosphate added to the 3 prime position of the ADP molecule, added to pantothenic acid, added to this short chain with the sulfur on the end. And this whole molecule together is coenzyme A. And so when we say acetyl-CoA, it's this giant molecule, S-thioester to the carbonyl, the acid on acetate, to CH3. And so that would be, basically, acetyl-CoA. And so one convenient thing about this is it's much easier than drawing that big molecule, but it is a little bit misleading in terms of its size. So, that's coenzyme A. Next one I want to talk about is FAD which stands for flavin adenine dinucleotide. So, flavin adenine dinucleotide is an electron carrier, just like NAD plus. And it's derived from the vitamin riboflavin, also referred to as vitamin B2. Another thing from the side of your cereal box, and basically looks like this. So, like NAD, it's a dinucleotide. And so here's ADP, just like we do for CoA, or just like one end of NAD. One difference is that the other nucleotide down here actually isn't technically a sugar. It's ribitol instead of ribose. What does that mean? It doesn't have an aldehyde. Instead, it is just a five-carbon chain where all of the carbons are alcohols. And so since there's no aldehyde, it doesn't form a ring. And the base on this end, as a nicotinamide, is this flavin group, which looks like this. And so this is FAD, which is in the oxidized form. Turns out, in the oxidized form, FAD is yellow, hence riboflavin-"flavin", yellow. And the reason it's yellow-- you see there's a conjugated double bond here across this part. It turns out this is the active part of the molecule, and it works as follows. And so if you have a hydride ion, remember the way we can transfer two electrons. Can transfer the two electrons that way, and that allows it to generate. And I'll just draw the middle, here. Active part of the molecule. So, that would be these two nitrogens. We've added two electrons to it. And so this is abbreviated FADH2, or the reduced form of FAD. And it is colorless, because now you no longer have that conjugated double bond system, loses its color. And so you can follow whether FAD is oxidized or reduced by just looking at color change. So, that's FAD. It's an electron carrier, carries electrons by a hydride transfer, very similarly to what we described for NAD, but obviously a different molecule.

~15:13And the last molecule, the last co-factor that we need for this reaction, is lipoic acid. Which, unlike most things in metabolism, doesn't have an abbreviation and also functions as an electron carrier. And so lipoic acid looks like this. So, that is lipoic acid. This is in the oxidized form. And so where it's oxidized is here at this sulfur-sulfur bond. So, you can think of this as the same as a disulfide bond that you learned about from Professor Yaffe in proteins. And so this is the oxidized form of the disulfide bond. If I take this hydride, transfer two electrons across that bond, then it goes to-- and I'll just draw here the active end. Then we go here to the reduced form of lipoic acid, and there's no abbreviation for oxidized or reduced lipoic acid. It's just lipoic acid, oxidized lipoic acid, reduced. And how they're oxidized and reduced is basically very similar to the disulfide bonds that you saw in proteins being oxidized, disulfide bond being oxidized, or it can be reduced to not be a disulfide bond. Now, it turns out that these cofactors, our TPP, FAD, and lipoic acid, are all stably associated with different subunits of a multi-protein complex that assembles to catalyze that reaction to convert pyruvate to acetyl-CoA. The enzyme or enzyme complex that carries this out is referred to as PDH, sometimes abbreviated PDC, which stands for the pyruvate dehydrogenase complex. So, pyruvate dehydrogenase, PDH, pyruvate dehydrogenase complex, PDC. This complex is basically three different polypeptides that come together to form a complex and catalyze that reaction. Now, where this complex sits-- so, this is the mitochondria. This is the matrix. That's where this pyruvate dehydrogenase reaction occurs. That's where the TCA cycle occurs. And this PDH complex is basically sitting here at the membrane on the matrix side of the membrane. Now, the three different polypeptides that come together to form this complex are creatively named E1, E2, and E3, four. Enzyme one, two, and three. And each of these, as I alluded to, is associated with a different co-factor. And so E1 is associated with thymine pyrophosphate. E2 is associated with lipoic acid. And E3 is associated with FAD. Now, let's go through the mechanism for how this pyruvate dehydrogenase reaction works. So remember, this is the active part of TBP plus. It is bound in the active site of E1. And it reacts with pyruvate to catalyze alpha decarboxylation, just like we described for conversion of pyruvate to alcohol. So, you're going to see exactly the same mechanism that we drew before. So, that decarboxylates the alpha-keto acid, just like we saw to generate acetaldehyde. The difference is rather than resolve this such that this carbon has the same oxidation state and make acetaldehyde, instead the next step of this reaction is going to be oxidized by reducing lipoic acid. So, the active part of lipoic acid that is in the active site of the E2 subunit. So this will now regenerate E1, but what we're left with, then, is this. Now, this intermediate bound to E2. Here's where coenzyme A can come in, which will then generate acetyl-CoA. But now we are left with E2 in the reduced state, rather than being in the oxidized state. So E2 has to be re-oxidized in order for this complex to carry out the next catalytic cycle, and the way that works is as follows.

~18:56So you have FAD bound on E3. And so you have a hydride ion from the oxidation of E3 that can be transferred to FAD. That will generate FAD from the oxidized to the reduced form. So re-oxidize lipoic acid on E2, reduce FAD to FADH2, and then that FADH2 can be re-oxidized back to FAD via transferring those electrons to NAD plus to generate NADH. So in this case, FADH2 re-oxidized the FAD, NAD plus reduced to NADH. And so, effectively, what this happens is that enzyme E1 and E2 cooperate to call what's referred to as oxidative alpha decarboxylation of pyruvate, while adding -CoA, so that's where you get acetyl-CoA, with reduction of lipoic acid in E2, and then E3 re-oxidizes the lipoic acid in E2 while generating NADH. This NADH, once it's generated, of course those electrons have to go somewhere, so they, like in glycolysis-- it also needs to regenerate to NAD at some point. This is ultimately the electrons that end up on oxygen, and it's really this series of electron transfers, with oxygen being a good electron acceptor, that ultimately allows controlled energy release during carbon oxidation. And cells, you'll see, can use that to make ATP or do other kinds of work. So, the net reaction and/or another way to draw the pyruvate dehydrogenase reaction would be as follows, and that's taking pyruvate to acetyl-CoA. And so we're going to take coenzyme A and release CO2. This is done by TPP plus, as part of E1. That involves converting the lipoic acid in E2 from the oxidized to the reduced state. That lipoic acid then has to be re-oxidized. If something's oxidized, something else has to be reduced. That's FAD on E3, which also then cycles between the oxidized and reduce state. And ultimately, those electrons ended up being transferred to NAD plus to generate NADH. And so the PDH complex is basically a chain of electron transfer reactions. And it's the first example of a chain of electron transfer reactions. We're going to see that there is the electron transport chain in the mitochondria, effectively does the same thing. And by coupling oxidation and reduction reactions across chains of molecules like this, effectively as a preview, allows the stepwise energy release of these oxidation reactions to occur. So remember, if we burn glucose, completely oxidize it in one step, where those electrons are directly transferred to oxygen in combustion. Lots of energy released, but all in one step. By doing these stepwise electron transfers, we can then basically break up that energy release in a way that can be captured by cells to do work. Here, the way energy is captured, it's not so obvious in this electron transport reaction. But effectively, you're using oxidation of the ketone on pyruvate, with decarboxylation to the acid to generate, rather than just the acid, a thioester bond. And that's that thioester bond-- as well as NADH, that's energy as well-- but it's really that thioester bond that then can be recaptured later to drive synthesis of citrate in the TCA cycle. Now, next what I want to do is I want to discuss the TCA cycle reactions. Now that you see how you can get acetyl-CoA, at least from pyruvate, I want to discuss how you can now use that acetyl-CoA and oxidize it back to combine it with oxaloacetate, and oxidize it back to make citrate, and then oxidize it back to oxaloacetate to run the TCA cycle. OK, great. So, the first reaction of the TCA cycle. Here's acetyl-CoA. It will combine with oxaloacetate. This reaction is catalyzed by an enzyme called citrate synthase, and generates the six-carbon tricarboxylic acid citrate. So, how does this reaction work?

~22:58Here's drawing acetyl-CoA in a slightly different way. If I redraw this as the enol, this will generate citrate via, effectively, that mechanism. Next, citrate is converted by adding water across this carbon-carbon, or by removing water across this carboncarbon bond, to generate this intermediate called cis-aconitate. So this intermediate is called cis-aconitate. so all I did was dehydrate across that bond so there's a double bond there. And then if I re-add water across that bond, I generate this molecule called isocitrate. So effectively, to convert citrate to isocitrate, I'm moving the hydroxyl group from that carbon to this carbon. To do that, I basically dehydrate, make a double bond, remove water, readd water across that bond in the opposite direction to generate this molecule, isocitrate. This reaction is carried out by an enzyme called aconitase, and converts citrate to isocitrate. I think it's a little easier to see this reaction if I draw it a slightly different way. So, this here is just drawing citrate by just slightly rotating the molecule to look like that. And so I'm basically removing water here. And then I'm now just adding water back in the opposite orientation to generate isocitrate. Now, you'll notice when I drew this-- if you look at citrate, this is actually a symmetrical molecule, so the top half and the bottom half of citrate are identical. And so what's interesting about nature is that it treats these carbons- the green carbons that came from acetyl-CoA-- different from the side of the molecule that comes from oxaloacetate. And effectively, nature always moves the hydroxyl group to this carbon that came from oxaloacetate, and never moves it to this carbon that came from acetyl-CoA. This is an example where enzymes-- nature treats a symmetrical molecule like citrate in an asymmetrical way. And this has consequences for how carbon is actually traced through the entire TCA cycle. Because even though you might think things could get scrambled at citrate, they never do. Meaning an isocitrate-- it's always these green carbons that came from acetyl-CoA. You never get those green carbons on the other side of isocitrate. And so when we go through the TCA cycle, I'll keep these carbons green until the point where you can no longer distinguish which carbon came from came from which reaction. The next reaction is we're going to oxidize this carbon of isocitrate. So if we're going to oxidize that carbon, those electrons have to go somewhere. And so if we oxidize the carbon, we can use NAD plus as an electron acceptor, reduce it to NADH. That generates this intermediate. So hopefully this is clear to everybody at this point, but just in case. So, this carbon here and isocitrate. If I oxidize that alcohol to the ketone, now I generate a hydride ion. Those two electrons and the hydrogen can go to NAD plus and reduce it to NADH. That generates this intermediate. It's called oxalosuccinate. Which then, if you notice, the oxalosuccinate is now a beta keto acid. So alpha, beta. The acid group is beta to the ketone, so it's a beta keto acid. Remember, beta decarboxylation is favorable, and so I can lose that CO2. And what I'm left with is this molecule, which is called alpha keto glutarate. So this whole reaction here, the oxidation of the alcohol to the ketone, followed by beta decarboxylation of oxalosuccinate to alpha ketoglutarate, is carried out by an enzyme called isocitrate dehydrogenase. And of course, just to remind you, here's that beta keto acid in oxalosuccinate. And so that can decarboxylate, leading this enol. Which can, of course, rearrange back to the ketone that we see, an alpha ketoglutarate.

~26:57Now, if you look at alpha ketoglutarate, you'll notice that-- and I will redraw over here. So, this here is just me redrawing alpha ketoglutarate, which is often abbreviated alpha kG. Just drew it as a straight line. Now, if you'll notice, alpha keto glutarate is a alpha ketoacid. And so here the acid group is alpha to the ketone. So, an alpha keto acid. It's effectively just like pyruvate, but has this additional pieces on it. And it turns out the next step in the TCA cycle is the exact reaction that we saw with pyruvate dehydrogenase. It's alpha decarboxylation, oxidative alpha keto acid decarboxylation, as follows. So, we carry out. This is a molecule called succinyl-CoA. And so, you'll see what happened there is decarboxylated here, this carbon, while oxidizing this ketone to the acid and adding -CoA. That's a redox reaction. So NAD, NADH, it turns out this is exactly the same mechanism-- so I don't need to draw it again-- that I just showed you for pyruvate dehydrogenase. So it needs all the same cofactors-- TPP plus, lipoic acid, FAD. And in fact, it even shares some of the same enzyme complexes, subunits, as pyruvate dehydrogenase. And so this is a reaction that's catalyzed by alpha ketoglutarate dehydrogenase. Like pyruvate dehydrogenase, this is a complex, and so it has a unique E1, which makes sense. Remember, E1 of pyruvate dehydrogenase was actually the subunit that bound the pyruvate. E1 of alpha ketoglutarate dehydrogenase is unique. It binds alpha ketoglutarate instead of pyruvate. However, they share the same E2 and E3s, and so the E2s and E3s would carry out exactly the same reaction and play the same part in the mechanism of how you do this alpha oxidative alpha decarboxylation to turn alpha ketoglutarate into succinyl-CoA. Now, remember in pyruvate dehydrogenase, once we got that acetyl-CoA, we then use this CoA group to drive condensation with citrate. Well, in this case, what happens is you don't want to combine condensation here. Instead, what is going to happen is you want to use this CoA group to now generate ATP. And so this is going to couple release of the CoA to generate an ATP equivalent. It's actually GTP that's generated by the TCA cycle. And so, this is going to generate this molecule, succinate. And the enzyme that does this is called succinic bio-kinase. So, let's go through over here how this enzyme works. So here's succinyl-CoA, basically using favorable loss of the CoA breaking the thioester bond to generate this acid anhydride. We saw an acid anhydride before in glycolysis. Remember, we made 1-3-bisphosphoglycerate, so that's a good phosphate donor. And then that can be used to generate succinate, and transferring that phosphate to GDP to make GTP, just like 1-3bisphosphoglycerate, was able to transfer the phosphate from the acid anhydride to ADP to make ATP in glycolysis. So, the next step is to oxidize this carbon-carbon bond in succinate. So if we're going to oxidize a carbon-carbon bond, those electrons have to go somewhere. So there's our carbon-carbon bond. And so if we generate hydride ion just like we did in other oxidation reactions, but this hydride ion is transferred not to NAD, but instead to FAD, a different electron carrier, to generate if FADH2. Now, I should point out succinate, like citrate, is a symmetrical molecule. But at this point, nature doesn't tell the difference. Once it generates succinate, this molecule now gets scrambled. And so everything downstream of succinate, you no longer know which carbons came from acetyl-CoA. This generates this molecule called fumarate, and this reaction is carried out by an enzyme called succinate dehydrogenase, often abbreviated SDH for succinate dehydrogenase.

~30:56Now, the next reaction is, we're going to add water across this double bond of fumarate. And that generates this intermediate, malate. This reaction is carried out by an enzyme called fumarate hydratase. And it's simply adding a water molecule across that double bond. Once we have malate, if you look what's the difference between malate and oxaloacetate, the difference is that in oxaloacetate, this carbon is a ketone. Whereas in malate, it's an alcohol. And so if we want to turn this carbon from the alcohol into the ketone, that is, of course, a oxidation reaction, and so those electrons have to go somewhere. Don't need to draw the mechanism again. It's basically just the hydride transfer to oxidize this to an alcohol, to the ketone. That oxidation couples to a reduction of NAD plus to NADH. This is carried out by an enzyme called malate dehydrogenase. And doing this completes the TCA cycle, regenerating oxaloacetate, that can then recombine with another acetyl-CoA to go through another round of the cycle. You'll notice that going through this cycle, there's two CO2s lost. One of them is lost here at the isocitrate to alpha ketoglutarate reaction. So, this decarboxylation from oxaloacetate to alpha ketoglutarate. That beta decarboxylation, that's the first CO2. The other one is lost here at the alpha ketoglutarate. The alpha ketoglutarate dehydrogenase step, where you have this alpha decarboxylation to take alpha ketoglutarate to succinyl-CoA oxidative alpha decarboxylation. Now, what's cool about this, as you noticed, we just discussed all the reactions of the TCA cycle. And I showed you, reminded you, of some chemistry that you've already seen, but unless you count the chemistry we showed you earlier for how the PDH and alpha ketoglutarate dehydrogenase reactions work with E1, E2 and E3, with lipoic acid, FAD. That was obviously new for today. But other than that, everything else was chemistry that you've already seen. And this really points out the point that I made earlier, that metabolism is really variations on relatively few reactions. We've just repurposed some of the same tricks, if you will, that we're used in glycolysis, and allowed it to now do an entire different pathway, the TCA cycle. It also points out how Hans Krebs was-- well, is still-remarkable, able to figure out from chemistry alone, because there's actually quite a bit of logic to the way metabolism works. Now, I want to say this again. Note there were two carbons that entered acetyl-CoA, and two carbons that were lost to CO2. But if you look, the green carbons remain in the same places until they get to succinate. and so the two carbons that enter are not lost on the first turn of the cycle. It's actually two carbons that came from oxaloacetate that are converted to CO2 as that acetyl-CoA goes through the cycle. And so to oxidize the exact carbons from acetyl-CoA to CO2 requires more than one turn of the cycle.

~34:07You'll also notice that the cycle is oxidation. And so oxidation reactions, of course, release energy. We've talked about that. And so it's favorable. And the products, if you will, are three NADH molecules. You can say plus one more NADH if we're going all the way from glucose or from pyruvate. Glucose derived pyruvate because the pyruvate dehydrogenase reaction also generates an NADH to make that a acetyl-CoA. One FADH2, as well as one GTP molecule. And so lots of oxidation going on here. We completely oxidized two carbons to CO2, so that's energy release. But you notice you only get one GTP from the molecule.

~34:49Now, this GTP, of course-- that reaction, the succinic thiokinase reaction, like the reactions we saw on glycolysis, is such that it can generate GTP at a high DGP-GDP ratio, or ATP-ADP ratio. Remember, those are equivalent. Those energy charge are similar, and so that makes sense. But most of the energy released is actually reducing NAD and FAD to NADH and FADH2. And of course, these need to transfer their electrons somewhere else, and that's the role of oxygen. Oxygen, remember, is a very good electron donor, and so it's the ultimate transfer of those electrons from these molecules to oxygen that also provide energy that the cell can use to do work, but it does so, in a way, by charging up different ratios in the cell. So the NAD-NADH or the FADH2-FAD ratios. And just like we talked about, the ratio or the energy of ATP is in the interconversion between ATP and ADP. It's the ratio that drives the free energy change. The same thing exists for an NADH and NAD, FADH2 and FAD. And so charging up these ratios while passing through the TCA cycle, and the ultimate downstream transfer of those electrons to oxygen, really is where most of the energy is captured as carbon is oxidized through the TCA cycle. And exactly how that works and how it can be related to ATP will be something that'll be more explicit in the coming lectures. Now, I want to point out apart from the oxidation, there's actually lots of intermediates made here. And it turns out, a bunch of these intermediates are useful for cells to make stuff. So we talked about gluconeogenesis. Gluconeogenesis needs electron balance. We get NADH from the TCA cycle, and so you can think of gluconeogenesis as an alternative to fermentation to dispose of electrons. Well, you can use the NADH from the TCA cycle to run gluconeogenesis as well. But beyond the cofactors, the carbon itself. So citrate, I've alluded to now a few times, is important as a precursor to make fat. We'll discuss that in later lectures, too. But other intermediates in this pathway are useful for various amino acids and nucleic acids. And so there's lots of things that can come from the TCA cycle that cells can find useful to do, not just catabolism, but also anabolic processes. Now, the way the TCA cycle works, though, is that there's actually an issue if you want to use the intermediates from the TCA cycle to make stuff. And so what is that issue? Well, the TCA cycle functions at a site as a cycle. And so, if we're going to take things in and out of it, that has consequences for how the cycle runs. Now there's a couple words for this that I want to just introduce to you. The first one is cataplerosis and the second one is anaplerosis. And so cataplerosis is the act of removing stuff from a metabolic cycle. So, we're going to remove citrate from the cycle to make fat. That's cataplerosis. And anaplerosis is adding stuff back to a metabolic cycle so it can continue to function. Viewing this is really evident if you think of the TCA cycle as a chicken and egg problem. So, the very first time acetyl-CoA was generated, how do you start the TCA cycle in the first place? You can't add it to the TCA cycle unless you have oxaloacetate to combine with the acetyl-CoA, which can then generate another oxaloacetate. So where does the first oxaloacetate come from? Well, we already talked about one reaction. We talked about it in the context of gluconeogenesis. That can solve this problem.

~38:47And so we have pyruvate. And so we talked earlier today how we can do oxidative decarboxylation of pyruvate to give acetyl-CoA. But we talked to in the gluconeogenesis lecture how we can add a CO2 pyruvate to generate oxaloacetate. So if I do those two reactions, now I have all the carbon I need to generate a citrate and start off the TCA cycle. Now obviously, if I do cataplerosis and I remove that citrate I made to make fat, well, now I need two pyruvate again to generate the next citrate if I'm using this pathway, because every time I bring an acetyl-CoA into the cycle, I need an oxaloacetate to combine it with. And so if I remove something, I have to add something back. And so pyruvate to oxaloacetate, the pyruvate carboxylase reaction is an example of an anaplerotic reaction. Now what this means, though, is that in order to do an anaplerosis, you have to be able to generate four-carbon oxaloacetate, or a four-carbon molecule. Now, pyruvate carboxylase, pyruvate to oxaloacetate, allows you to do that. We can take a three-carbon molecule and generate four-carbon oxaloacetate. However, if we start from a two-carbon molecule like acetate or acetyl-CoA that enters the cycle, it's actually not so simple to take that two-carbon molecule and turn it into four-carbon oxaloacetate. And in fact, humans lack any enzymes that allow them to take a two-carbon unit, to take acetate or acetyl-CoA, and turn it into anything that's longer than net-- turn it into anything that's longer than two carbons. And this has important implications for human physiology, because what it says is that we can't make glucose from anything that starts with something less than three carbons long. So if you drink alcohol and you metabolize that alcohol to acetate, it turns out you take fat and you break down fat also to acetyl-CoA, to acetate two carbons long. There is no way to turn those molecules into glucose, because you cannot generate the oxaloacetate to do the anaplerosis that's necessary to get it there. What this means is that our body can only store calories that come from two carbon units, fat or alcohol, as fat. We can never turn them back into glucose or make glycogen. And this is very relevant for those of you that go to medical school, because it's relevant to our physiology. And that is, when our bodies exhaust all of our stores of glucose, what happens? Our liver can no longer do gluconeogenesis. And so what happens? Now it has to switch over to doing something else. It has to work with two carbon units. And ultimately, this is ketone metabolism, which we'll talk about in a few lectures. It also said that the body-- that this is also the basis of a very old adage that's out there that some of you may have heard-- that you need to have some other fuel if you're going to burn fat. The basis for that is that fat is turned into two carbon units, acetyl-CoA units. And so if you're going to take those acetyl-CoA units and ultimately burn them away, turn them into CO2, you need a source of oxaloacetate, or your TCA cycle won't work. And you don't need a lot of something, but it is true. You can't start just with acetyl-CoA as a human and turn it into CO2, so you need some-- at least a little bit of oxaloacetate to get your TCA cycle started. Now, that's a problem that we as humans and other mammals face, but it turns out there's lots of microbes out there that grow just fine on acetate or on alcohol, even if it's only carbon source. And so those organisms must have some way to build stuff from two carbon units. That is a way to use two carbon units and do an anaplerosis. And it turns out the way they do this is via something called the glyoxylate cycle.

~43:06And so the glyoxylate cycle is an alternative version of the TCA cycle that effectively uses two enzymes that we lack as mammals. And so I'll quickly tell you about it here. So, this is isocitrate from the TCA cycle. And some microbes have an enzyme called isocitrate lyase. And what isocitrate lyase does is basically splits citrate in half, such that the top portion of the molecule is another TCA cycle intermediate, succinate. And the bottom portion of a molecule is this two carbon aldehyde called glyoxylate. Glyoxylate can react with acetyl-CoA by another enzyme that we lack as humans called malate synthase. And I don't have time to show the mechanism again, but malate synthase basically adds the two carbons from acetyl-CoA to the aldehyde, the carbonyl, the aldehyde carbonyl of glyoxylate in a reaction that is, for all intents and purposes, exactly what happens in citrate synthase. That will generate this molecule, which is malate, also in the TCA cycle. And so having these two extra reactions, isocitrate lyase and malate synthase, gives microbes the ability to have acetyl-CoA be anaplerotic. And so how does that work? Well, that's because if we start with one oxaloacetate, four carbons, and acetyl-CoA, two carbons, can run the TCA cycle and make citrate, six carbons. Turn that citrate into isocitrate. Use isocitrate lyase to generate glyoxylate, two carbons. Plus succinate. That succinate can run through succinate dehydrogenase to generate malate, which you can go through malate dehydrogenase to generate oxaloacetate. This glyoxylate can start with a second acetyl-CoA. Two carbons come together, generate malate. That generates a second malate molecule, which can then exit the cycle as malate or oxaloacetate or whatever you want. And so basically, it allows two acetyl-CoAs to net generate an oxaloacetate, and so net generates a way to do an anaplerosis from two carbon units by having this malate synthase reaction and this isocitrate lyase reaction and run this alternative version of the TCA cycle, called the glyoxylate cycle. And it's a nice way how life-- again, no new chemistry here, just variations on what we've already shown-- repurposed the similar chemistries that it's already using as a way to live off of carbon sources that contain only two carbons, like ethanol or acetate. So in closing today, the last thing I want to talk about, very briefly, is how the TCA cycle is regulated. And we don't need to spend a ton of time on this, because it really follows principles that make sense, particularly when we think about things that we've already talk about. And so, regulation of the TCA cycles is of course important, and it's really a critical hub, both for anabolic and catabolic pathways. So, you needed to get energy to fully oxidize carbon, but it's also a useful place to get stuff. And before I talk about regulation, I just want to point out that many of the enzymes in the TCA cycle, even though we talk about it in the mitochondria and we're about to talk about regulation in terms of catabolism-- that is oxidation, ways to release energy-- many of these enzymes are also in other locations in the cell, because there's functions for them in making stuff that is very different than what goes on in the TCA cycle. And the regulation that I'll tell you about, and that comes up on MCAT exams and stuff like that, usually talks about this pathway as a catabolic pathway, as a way to make CO2, to make ATP. However, recognize that there's also variations on this pathway that can use in anabolic things, making stuff. And that regulation is something that really is not necessarily what we're going to talk about here and is a little bit less well-understood.

~47:10But at least the regulation, in terms of catabolism, if we put it in context of glucose metabolism-- so here's glycolysis, turning glucose into pyruvate. And then that pyruvate can operate through the pyruvate dehydrogenase reaction to generate acetyl-CoA. That acetyl-CoA can combine with oxaloacetate to generate citrate. That citrate, of course, can be used to generate fat, as we've talked about. That can go to isocitrate. Isocitrate to alpha ketoglutarate. That's catalyzed by isocitrate dehydrogenase, which I will abbreviate IDH. Alpha ketoglutarate to succinate. More correctly, to succinyl-CoA by alpha ketoglutarate dehydrogenase, Alpha KGDH. And then this back to oxaloacetate. And so the main enzymes that are typically discussed as being regulated are alpha ketoglutarate dehydrogenase, isocitrate dehydrogenase, and pyruvate dehydrogenase. I also drew glycolysis. And I'll just write over here, gluconeogenesis up here, because we can now see more fully how this plays in with citrate acting as a positive regulator with gluconeogenesis and an inhibitor of glycolysis. Again, we're full in citrate, stop running glycolysis, start running gluconeogenesis. Now, the other regulation. If you have a lot of acetyl-CoA, stop making it from pyruvate. acetyl-CoA is a negative regulator of PDH. This is carbon oxidation. Releases a lot of energy, generates a lot of ATP, generates a lot of NADH. If you have lots of those things, no reason to keep sending carbon into acetyl-CoA to go into the TCA cycle. Ultimately, we have to send those electrons somewhere. So if there's nowhere to put them, low oxygen also inhibits pyruvate dehydrogenase. And of course, if you need more energy, if ADP is high, that activates pyruvate dehydrogenase. So at least in terms of glucose, complete glucose oxidation, a lot of regulation happens at pyruvate dehydrogenase. And a lot of it really makes sense. High levels of ATP, high levels of NADH, also inhibit isocitrate dehydrogenase and alpha ketoglutarate dehydrogenase. Makes sense. Succinol-CoA high, inhibit alpha ketoglutarate dehydrogenase. High levels of ADP, need to release more energy, activate alpha ketoglutarate dehydrogenase. Again, these are the feedbacks that people talk about on board exams. Good to know, but you can almost guess what they would be from first principles. Because remember, this is a pathway that releases a lot of energy. High energy high ATP, high NADH-- don't run the cycle, don't enter carbon in the cycle. Low energy, high ADP, put carbon in the cycle, run the cycle faster. Makes sense. Also makes sense of the reciprocal regulation of glycolysis and gluconeogenesis. Great. Next time, we will talk more about how we can oxidize fatty acids, fat, by accessing acetyl-CoA and entering it into the cycle. Thanks.

3.5同位素追踪之谜与能量总账：为什么第一轮脱掉的 CO₂ 100% 来自草酰乙酸？

一、MIT 7.05 L23 压轴推演：TCA 循环 ¹⁴C 同位素追踪实验与前手性识别

在 L23 课堂上，Vander Heiden 教授特地拿起绿色粉笔，把从乙酰-CoA 进入柠檬酸的那两个碳原子涂成绿色，向全班展示了一桩曾让 20 世纪 40 年代顶尖生物化学家（包括 Krebs 本人！）差点否定柠檬酸是 TCA 循环中间物的历史悬案。这不仅是 MIT 7.05 期中考试的压轴大题，更是国内名校生化考研同位素追踪题的母题！

二、1 分子葡萄糖彻底有氧氧化的能量总账（30/32 ATP 新旧口径完美对照）

现在，我们已经集齐了葡萄糖有氧氧化的三大阶段：糖酵解（细胞质） → 丙酮酸氧化脱羧（线粒体基质） → 柠檬酸循环（线粒体基质 + 内膜）。当这些阶段产生的全部 NADH 和 FADH₂ 进入线粒体内膜呼吸链完成氧化磷酸化时，1 分子葡萄糖究竟能换来多少分子 ATP？这是华农生化考研计算题与简答题的“镇卷之宝”！

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🎧 MIT 7.05 课堂原声英文实录对照（本节关联 1 讲 · 完整逐段实录）点击展开 / 收起 ▾

🎬 MIT 7.05 Lec 23 · TCA Cycle II, Isotope Tracing & Glyoxylate Cycle⏱ ~50:00

~00:00MITOCW | 23. TCA Cycle II

~00:02MATTHEW

~00:02Hello, everybody. Last time, I introduced the idea of the TCA cycle, tricarboxylic acid cycle. Also known as the

~00:10VANDER

~00:10citric acid cycle, because citric acid is a tricarboxylic acid, as you'll see later today. Also known as the Krebs

~00:18HEIDEN:

~00:18cycle, named after Hans Krebs, who discovered it in the early part of the last century. The TCA cycle is the series of reactions that occurs in the mitochondrial matrix and it allows the complete oxidation of two carbon units, derived from many things, including pyruvate, derived from glucose and glycolysis and enables the complete oxidation of that carbon to CO2. Now, it's a cycle because those two carbon units from pyruvate or other sources enter the cycle, combine with 4carbon oxaloacetate, and combine to make 6-carbon citrate. Hence the citric acid cycle, or TCA cycle, that is then oxidized back to 4 carbon units, forming a cycle that allows cells to release lots of energy. Complete oxygen enables, ultimately, complete oxidation of glucose to CO2. We've talked many times how this releases energy. And it also generates lots of intermediates for the cell that can be used to make other stuff. Last time, I alluded to the fact that citrate can be used, say, to make fatty acids. Today, what I want to do is I want to dive into the details of how this TCA cycle occurs and the consequences of the way it works, and how that affects other aspects of metabolism. You'll see that it actually affects the ability of different organisms, whether or not they can make things from intermediates in this cycle, because of how the cycle works. Now, to start. Of course, if we're going to start from pyruvate. Pyruvate, of course, has three carbons. And so if we're going to generate a two-carbon acetate group, we have to lose CO2. And as I described a couple lectures ago, acetate is also the thing that we can derive from metabolism of ethanol, showing us two things-- metabolized glucose to pyruvate or ethanol itself-- can both be turned into acetate. But really, the donor is this molecule, acetyl-CoA. Which is basically also a carboxylic acid, but rather than being a carboxylic acid, instead makes this a thioester bond, which activates this carbon as a leaving group, such that it can combine with oxaloacetate, which you'll remember from gluconeogenesis, releasing that S-CoA molecule to generate six-carbon citric. So, this is citric. You can see that we've made a bond from this carbon on the acetate, losing this S-CoA group to this carbon oxaloacetate to make this six-carbon citrate molecule, which is a one, two, three-- three-carboxylic acid, or tricarboxylic acid. Hence the name citric acid cycle, tricarboxylic acid cycle.

~02:58Now, these six carbons can then-- or this six-carbon citrate molecule can then be oxidized, generating two CO2 molecules that are released, and ultimately reforming oxaloacetate that can pick up another two-carbon acetylCoA to generate another citrate, and around and around the cycle goes, allowing in the end the net entry of two carbons, effectively from acetate, and release of two carbons as CO2. Now, as I alluded to, this can come from pyruvate. It can come from acetate itself, vinegar. It can come from alcohol. It turns out that when you break down fat, you also break it into two carbon units. And so this cycle becomes very useful for cells, because it allows the oxidation of many different molecules to completely turn that carbon into CO2. Now, if we're going to do this from glucose, however, you'll remember that pyruvate has three carbons. And so if we're going to turn pyruvate into acetate, or acetyl-CoA, we have to lose a carbon of CO2. We have to lose this carbon as CO2. Now, we saw this before, that we can do this via-- this is exactly how we generated ethanol when we did fermentation of pyruvate to ethanol, but that molecule didn't generate acetate, It generated acetaldehyde. The difference, of course, being whether or not this carbon gets oxidized in the case of acetate to the acid, or in acetaldehyde in ethanol metabolism, where it remains an aldehyde. Now obviously, you can make ethanol and then turn that ethanol into acetate. And that's a pathway that I guess certainly would work. However, that's not the way it happens in most organisms. Most organisms actually directly produce acetate, or more correctly, acetyl-CoA from pyruvate. So let's take a look at that reaction. So here once again is pyruvate. And if we turn that pyruvate into this two-carbon acetyl-CoA, let's look here what has to happen. Well, the first thing is that we have to decarboxylate this molecule. And so that generates a CO2. We have to lose that one carbon. Also, as I pointed out, if we do this as if we did it in ethanol metabolism, we'd be left with an aldehyde. But this is an acid, and so this carbon also has to be oxidized. We know how to do that. We can donate those electrons to something like NAD, so the NAD is reduced to NADH. And we had to add this S-CoA molecule, which I'll come to in a minute. And so each of these steps ends up making a fairly complicated reaction. Obviously, several co-factors are going to be involved. You should be able to guess that just by looking at it. Obviously, I already drew up there NAD. If we're going to decarboxylate, remember this is a alpha carboxylic, an alpha ketoacid-- the ketone group is alpha to the carboxylic acid. And so if we're doing alpha decarboxylation, as you might guess, we need a cofactor. That co-factor, as I told you before, is the co-factor factor thiamine pyrophosphate. And finally, there's this S-CoA group. We need to describe what that is. However, before delving into those, and there's actually a couple other factors that are needed as well, I want to mention one other issue about this reaction, in that this reaction happens in the mitochondria, because that's also where the TCA cycle happens. So, recall that if we divide the cell into two compartments like an eukaryotic cell, here we have the cytosol and the mitochondria in a eukaryotic cell. As we've already talked about, having different compartments helps facilitate different metabolic reactions, because you can have different conditions in the two compartments.

~06:54And so as we said, glycolysis occurs in the cytosol. Glucose to pyruvate. And last time I mentioned, the TCA cycle occurs in the mitochondria. And so that means if we're going to fully oxidize the pyruvate carbon to CO2 using the TCA cycle in the mitochondria, that pyruvate has to get from the cytosol to the mitochondria, or at least carbons from the pyruvate have to get there. It turns out, you'll see in a minute, acetyl-CoA is a very large group. And so pyruvate itself is transported into the mitochondria. And that's where the reaction occurs to turn it into acetyl-CoA that can then enter the TCA cycle and oxidize. However, what this means is that a transporter is actually needed to get this across the mitochondrial membranes and into the matrix of the mitochondria where the TCA cycle happens. And I like to mention this because it turns out the pyruvate carrier-- that is, the transporter-- the way that it actually gets that pyruvate from the cytosol into the mitochondria actually was an unknown thing about metabolism until 2012, so not that long ago. Sometimes one can be left with the feeling, listening to these metabolism classes or reading a textbook, that everything about metabolism has been known forever, but it's actually not true. Here's a very key, central part of the pathway that was actually just discovered, at least at the time of this lecture, less than 10 years ago. It also illustrates that not all metabolism is completely understood. Now, let's get back to this reaction, how you turn pyruvate into acetyl-CoA. And I want to go through now and point out that several co-factors are needed. And so I already mentioned one of them, S-CoA, which is shorthand for coenzyme A. So, I need to tell you what that is. You hopefully are already familiar with TPP plus, thiamine pyrophosphate. We talked about that when we talked about how you do alpha decarboxylation to generate ethanol from pyruvate, also used here for the alpha decarboxylation reaction. Redox reaction happens, and so we needed NAD plus to get converted into NADH. We talked a lot about how that serves as an electron carrier, but it turns out that there's two additional electron carriers that are involved in this reaction. One of them is called FAD and the other one is called lipoic acid. Now you might say, why do we need all these electron carriers? Well, these are just different molecules that can carry two electrons, similar to NADH. And effectively, what these can do by having multiple electron carriers-- one can build chains of oxidation and reduction reactions. And it turns out these chains of oxidation reduction reactions really become central to energy transfers in biology, because building these chains allows more easy stepwise release of energy as one moves across these oxidation reduction reactions. Which remember, as I alluded to earlier, really are at the core of bioenergetics and a lot of what allows energy release from these pathways. What I mean by this will be more explicit as we go through what some of these co-factors look like. So, I'm not going to draw TPP plus or NAD again, but let's define what some of these other cofactors look like. So, let's start with coenzyme A. So, coenzyme A, it turns out, is useful. It's actually involved in lots of acylation reactions.

~10:34What's an acylation reaction? Well, that's basically if you're making a carbon-carbon bond by adding a molecule of greater than one carbon, so two carbons or greater, to something else. That's an acylation reaction, as we did with adding the two-carbon acetate to oxaloacetate to make citrate. And you'll actually see coenzyme A will come up in this in many, many lectures throughout the rest of the course.

~11:00Why this becomes useful is because it activates this basically carbon to the left of, in this case, the ketone. Because, by having that thioester there, ends up activating that carbon and allows it to carry out these acylation reactions. Coenzyme A itself is derived from a vitamin called pantothenic acid. And as a vitamin, this is something that you have to get from the diet. Now, when we abbreviate coenzyme A, which is often abbreviated S-CoA, you get the sense that it's this little tiny molecule that's just stuck on sulfur-- stuck on to the end to make this thioester bond. But it turns out coenzyme A is actually a giant molecule, one of the reasons why you actually synthesize acetyl-CoA in the mitochondria, because it's hard to transport this giant molecule. And so this is what pantothenic acid looks like. So, if I put an acid group here and a hydroxyl group there, that would be pantothenic acid, the thing that's in your cereal, on the side of your cereal box. Then there's these additional pieces. This is the active end of the molecule, that's that sulfur from the S-CoA, and then this side of the molecule is esterified to two phosphates, which are esterified to-- I'm not going to draw it out, but this would have an adenine base and a phosphate there. So basically, this is ADP, with a phosphate added to the 3 prime position of the ADP molecule, added to pantothenic acid, added to this short chain with the sulfur on the end. And this whole molecule together is coenzyme A. And so when we say acetyl-CoA, it's this giant molecule, S-thioester to the carbonyl, the acid on acetate, to CH3. And so that would be, basically, acetyl-CoA. And so one convenient thing about this is it's much easier than drawing that big molecule, but it is a little bit misleading in terms of its size. So, that's coenzyme A. Next one I want to talk about is FAD which stands for flavin adenine dinucleotide. So, flavin adenine dinucleotide is an electron carrier, just like NAD plus. And it's derived from the vitamin riboflavin, also referred to as vitamin B2. Another thing from the side of your cereal box, and basically looks like this. So, like NAD, it's a dinucleotide. And so here's ADP, just like we do for CoA, or just like one end of NAD. One difference is that the other nucleotide down here actually isn't technically a sugar. It's ribitol instead of ribose. What does that mean? It doesn't have an aldehyde. Instead, it is just a five-carbon chain where all of the carbons are alcohols. And so since there's no aldehyde, it doesn't form a ring. And the base on this end, as a nicotinamide, is this flavin group, which looks like this. And so this is FAD, which is in the oxidized form. Turns out, in the oxidized form, FAD is yellow, hence riboflavin-"flavin", yellow. And the reason it's yellow-- you see there's a conjugated double bond here across this part. It turns out this is the active part of the molecule, and it works as follows. And so if you have a hydride ion, remember the way we can transfer two electrons. Can transfer the two electrons that way, and that allows it to generate. And I'll just draw the middle, here. Active part of the molecule. So, that would be these two nitrogens. We've added two electrons to it. And so this is abbreviated FADH2, or the reduced form of FAD. And it is colorless, because now you no longer have that conjugated double bond system, loses its color. And so you can follow whether FAD is oxidized or reduced by just looking at color change. So, that's FAD. It's an electron carrier, carries electrons by a hydride transfer, very similarly to what we described for NAD, but obviously a different molecule.

~15:13And the last molecule, the last co-factor that we need for this reaction, is lipoic acid. Which, unlike most things in metabolism, doesn't have an abbreviation and also functions as an electron carrier. And so lipoic acid looks like this. So, that is lipoic acid. This is in the oxidized form. And so where it's oxidized is here at this sulfur-sulfur bond. So, you can think of this as the same as a disulfide bond that you learned about from Professor Yaffe in proteins. And so this is the oxidized form of the disulfide bond. If I take this hydride, transfer two electrons across that bond, then it goes to-- and I'll just draw here the active end. Then we go here to the reduced form of lipoic acid, and there's no abbreviation for oxidized or reduced lipoic acid. It's just lipoic acid, oxidized lipoic acid, reduced. And how they're oxidized and reduced is basically very similar to the disulfide bonds that you saw in proteins being oxidized, disulfide bond being oxidized, or it can be reduced to not be a disulfide bond. Now, it turns out that these cofactors, our TPP, FAD, and lipoic acid, are all stably associated with different subunits of a multi-protein complex that assembles to catalyze that reaction to convert pyruvate to acetyl-CoA. The enzyme or enzyme complex that carries this out is referred to as PDH, sometimes abbreviated PDC, which stands for the pyruvate dehydrogenase complex. So, pyruvate dehydrogenase, PDH, pyruvate dehydrogenase complex, PDC. This complex is basically three different polypeptides that come together to form a complex and catalyze that reaction. Now, where this complex sits-- so, this is the mitochondria. This is the matrix. That's where this pyruvate dehydrogenase reaction occurs. That's where the TCA cycle occurs. And this PDH complex is basically sitting here at the membrane on the matrix side of the membrane. Now, the three different polypeptides that come together to form this complex are creatively named E1, E2, and E3, four. Enzyme one, two, and three. And each of these, as I alluded to, is associated with a different co-factor. And so E1 is associated with thymine pyrophosphate. E2 is associated with lipoic acid. And E3 is associated with FAD. Now, let's go through the mechanism for how this pyruvate dehydrogenase reaction works. So remember, this is the active part of TBP plus. It is bound in the active site of E1. And it reacts with pyruvate to catalyze alpha decarboxylation, just like we described for conversion of pyruvate to alcohol. So, you're going to see exactly the same mechanism that we drew before. So, that decarboxylates the alpha-keto acid, just like we saw to generate acetaldehyde. The difference is rather than resolve this such that this carbon has the same oxidation state and make acetaldehyde, instead the next step of this reaction is going to be oxidized by reducing lipoic acid. So, the active part of lipoic acid that is in the active site of the E2 subunit. So this will now regenerate E1, but what we're left with, then, is this. Now, this intermediate bound to E2. Here's where coenzyme A can come in, which will then generate acetyl-CoA. But now we are left with E2 in the reduced state, rather than being in the oxidized state. So E2 has to be re-oxidized in order for this complex to carry out the next catalytic cycle, and the way that works is as follows.

~18:56So you have FAD bound on E3. And so you have a hydride ion from the oxidation of E3 that can be transferred to FAD. That will generate FAD from the oxidized to the reduced form. So re-oxidize lipoic acid on E2, reduce FAD to FADH2, and then that FADH2 can be re-oxidized back to FAD via transferring those electrons to NAD plus to generate NADH. So in this case, FADH2 re-oxidized the FAD, NAD plus reduced to NADH. And so, effectively, what this happens is that enzyme E1 and E2 cooperate to call what's referred to as oxidative alpha decarboxylation of pyruvate, while adding -CoA, so that's where you get acetyl-CoA, with reduction of lipoic acid in E2, and then E3 re-oxidizes the lipoic acid in E2 while generating NADH. This NADH, once it's generated, of course those electrons have to go somewhere, so they, like in glycolysis-- it also needs to regenerate to NAD at some point. This is ultimately the electrons that end up on oxygen, and it's really this series of electron transfers, with oxygen being a good electron acceptor, that ultimately allows controlled energy release during carbon oxidation. And cells, you'll see, can use that to make ATP or do other kinds of work. So, the net reaction and/or another way to draw the pyruvate dehydrogenase reaction would be as follows, and that's taking pyruvate to acetyl-CoA. And so we're going to take coenzyme A and release CO2. This is done by TPP plus, as part of E1. That involves converting the lipoic acid in E2 from the oxidized to the reduced state. That lipoic acid then has to be re-oxidized. If something's oxidized, something else has to be reduced. That's FAD on E3, which also then cycles between the oxidized and reduce state. And ultimately, those electrons ended up being transferred to NAD plus to generate NADH. And so the PDH complex is basically a chain of electron transfer reactions. And it's the first example of a chain of electron transfer reactions. We're going to see that there is the electron transport chain in the mitochondria, effectively does the same thing. And by coupling oxidation and reduction reactions across chains of molecules like this, effectively as a preview, allows the stepwise energy release of these oxidation reactions to occur. So remember, if we burn glucose, completely oxidize it in one step, where those electrons are directly transferred to oxygen in combustion. Lots of energy released, but all in one step. By doing these stepwise electron transfers, we can then basically break up that energy release in a way that can be captured by cells to do work. Here, the way energy is captured, it's not so obvious in this electron transport reaction. But effectively, you're using oxidation of the ketone on pyruvate, with decarboxylation to the acid to generate, rather than just the acid, a thioester bond. And that's that thioester bond-- as well as NADH, that's energy as well-- but it's really that thioester bond that then can be recaptured later to drive synthesis of citrate in the TCA cycle. Now, next what I want to do is I want to discuss the TCA cycle reactions. Now that you see how you can get acetyl-CoA, at least from pyruvate, I want to discuss how you can now use that acetyl-CoA and oxidize it back to combine it with oxaloacetate, and oxidize it back to make citrate, and then oxidize it back to oxaloacetate to run the TCA cycle. OK, great. So, the first reaction of the TCA cycle. Here's acetyl-CoA. It will combine with oxaloacetate. This reaction is catalyzed by an enzyme called citrate synthase, and generates the six-carbon tricarboxylic acid citrate. So, how does this reaction work?

~22:58Here's drawing acetyl-CoA in a slightly different way. If I redraw this as the enol, this will generate citrate via, effectively, that mechanism. Next, citrate is converted by adding water across this carbon-carbon, or by removing water across this carboncarbon bond, to generate this intermediate called cis-aconitate. So this intermediate is called cis-aconitate. so all I did was dehydrate across that bond so there's a double bond there. And then if I re-add water across that bond, I generate this molecule called isocitrate. So effectively, to convert citrate to isocitrate, I'm moving the hydroxyl group from that carbon to this carbon. To do that, I basically dehydrate, make a double bond, remove water, readd water across that bond in the opposite direction to generate this molecule, isocitrate. This reaction is carried out by an enzyme called aconitase, and converts citrate to isocitrate. I think it's a little easier to see this reaction if I draw it a slightly different way. So, this here is just drawing citrate by just slightly rotating the molecule to look like that. And so I'm basically removing water here. And then I'm now just adding water back in the opposite orientation to generate isocitrate. Now, you'll notice when I drew this-- if you look at citrate, this is actually a symmetrical molecule, so the top half and the bottom half of citrate are identical. And so what's interesting about nature is that it treats these carbons- the green carbons that came from acetyl-CoA-- different from the side of the molecule that comes from oxaloacetate. And effectively, nature always moves the hydroxyl group to this carbon that came from oxaloacetate, and never moves it to this carbon that came from acetyl-CoA. This is an example where enzymes-- nature treats a symmetrical molecule like citrate in an asymmetrical way. And this has consequences for how carbon is actually traced through the entire TCA cycle. Because even though you might think things could get scrambled at citrate, they never do. Meaning an isocitrate-- it's always these green carbons that came from acetyl-CoA. You never get those green carbons on the other side of isocitrate. And so when we go through the TCA cycle, I'll keep these carbons green until the point where you can no longer distinguish which carbon came from came from which reaction. The next reaction is we're going to oxidize this carbon of isocitrate. So if we're going to oxidize that carbon, those electrons have to go somewhere. And so if we oxidize the carbon, we can use NAD plus as an electron acceptor, reduce it to NADH. That generates this intermediate. So hopefully this is clear to everybody at this point, but just in case. So, this carbon here and isocitrate. If I oxidize that alcohol to the ketone, now I generate a hydride ion. Those two electrons and the hydrogen can go to NAD plus and reduce it to NADH. That generates this intermediate. It's called oxalosuccinate. Which then, if you notice, the oxalosuccinate is now a beta keto acid. So alpha, beta. The acid group is beta to the ketone, so it's a beta keto acid. Remember, beta decarboxylation is favorable, and so I can lose that CO2. And what I'm left with is this molecule, which is called alpha keto glutarate. So this whole reaction here, the oxidation of the alcohol to the ketone, followed by beta decarboxylation of oxalosuccinate to alpha ketoglutarate, is carried out by an enzyme called isocitrate dehydrogenase. And of course, just to remind you, here's that beta keto acid in oxalosuccinate. And so that can decarboxylate, leading this enol. Which can, of course, rearrange back to the ketone that we see, an alpha ketoglutarate.

~26:57Now, if you look at alpha ketoglutarate, you'll notice that-- and I will redraw over here. So, this here is just me redrawing alpha ketoglutarate, which is often abbreviated alpha kG. Just drew it as a straight line. Now, if you'll notice, alpha keto glutarate is a alpha ketoacid. And so here the acid group is alpha to the ketone. So, an alpha keto acid. It's effectively just like pyruvate, but has this additional pieces on it. And it turns out the next step in the TCA cycle is the exact reaction that we saw with pyruvate dehydrogenase. It's alpha decarboxylation, oxidative alpha keto acid decarboxylation, as follows. So, we carry out. This is a molecule called succinyl-CoA. And so, you'll see what happened there is decarboxylated here, this carbon, while oxidizing this ketone to the acid and adding -CoA. That's a redox reaction. So NAD, NADH, it turns out this is exactly the same mechanism-- so I don't need to draw it again-- that I just showed you for pyruvate dehydrogenase. So it needs all the same cofactors-- TPP plus, lipoic acid, FAD. And in fact, it even shares some of the same enzyme complexes, subunits, as pyruvate dehydrogenase. And so this is a reaction that's catalyzed by alpha ketoglutarate dehydrogenase. Like pyruvate dehydrogenase, this is a complex, and so it has a unique E1, which makes sense. Remember, E1 of pyruvate dehydrogenase was actually the subunit that bound the pyruvate. E1 of alpha ketoglutarate dehydrogenase is unique. It binds alpha ketoglutarate instead of pyruvate. However, they share the same E2 and E3s, and so the E2s and E3s would carry out exactly the same reaction and play the same part in the mechanism of how you do this alpha oxidative alpha decarboxylation to turn alpha ketoglutarate into succinyl-CoA. Now, remember in pyruvate dehydrogenase, once we got that acetyl-CoA, we then use this CoA group to drive condensation with citrate. Well, in this case, what happens is you don't want to combine condensation here. Instead, what is going to happen is you want to use this CoA group to now generate ATP. And so this is going to couple release of the CoA to generate an ATP equivalent. It's actually GTP that's generated by the TCA cycle. And so, this is going to generate this molecule, succinate. And the enzyme that does this is called succinic bio-kinase. So, let's go through over here how this enzyme works. So here's succinyl-CoA, basically using favorable loss of the CoA breaking the thioester bond to generate this acid anhydride. We saw an acid anhydride before in glycolysis. Remember, we made 1-3-bisphosphoglycerate, so that's a good phosphate donor. And then that can be used to generate succinate, and transferring that phosphate to GDP to make GTP, just like 1-3bisphosphoglycerate, was able to transfer the phosphate from the acid anhydride to ADP to make ATP in glycolysis. So, the next step is to oxidize this carbon-carbon bond in succinate. So if we're going to oxidize a carbon-carbon bond, those electrons have to go somewhere. So there's our carbon-carbon bond. And so if we generate hydride ion just like we did in other oxidation reactions, but this hydride ion is transferred not to NAD, but instead to FAD, a different electron carrier, to generate if FADH2. Now, I should point out succinate, like citrate, is a symmetrical molecule. But at this point, nature doesn't tell the difference. Once it generates succinate, this molecule now gets scrambled. And so everything downstream of succinate, you no longer know which carbons came from acetyl-CoA. This generates this molecule called fumarate, and this reaction is carried out by an enzyme called succinate dehydrogenase, often abbreviated SDH for succinate dehydrogenase.

~30:56Now, the next reaction is, we're going to add water across this double bond of fumarate. And that generates this intermediate, malate. This reaction is carried out by an enzyme called fumarate hydratase. And it's simply adding a water molecule across that double bond. Once we have malate, if you look what's the difference between malate and oxaloacetate, the difference is that in oxaloacetate, this carbon is a ketone. Whereas in malate, it's an alcohol. And so if we want to turn this carbon from the alcohol into the ketone, that is, of course, a oxidation reaction, and so those electrons have to go somewhere. Don't need to draw the mechanism again. It's basically just the hydride transfer to oxidize this to an alcohol, to the ketone. That oxidation couples to a reduction of NAD plus to NADH. This is carried out by an enzyme called malate dehydrogenase. And doing this completes the TCA cycle, regenerating oxaloacetate, that can then recombine with another acetyl-CoA to go through another round of the cycle. You'll notice that going through this cycle, there's two CO2s lost. One of them is lost here at the isocitrate to alpha ketoglutarate reaction. So, this decarboxylation from oxaloacetate to alpha ketoglutarate. That beta decarboxylation, that's the first CO2. The other one is lost here at the alpha ketoglutarate. The alpha ketoglutarate dehydrogenase step, where you have this alpha decarboxylation to take alpha ketoglutarate to succinyl-CoA oxidative alpha decarboxylation. Now, what's cool about this, as you noticed, we just discussed all the reactions of the TCA cycle. And I showed you, reminded you, of some chemistry that you've already seen, but unless you count the chemistry we showed you earlier for how the PDH and alpha ketoglutarate dehydrogenase reactions work with E1, E2 and E3, with lipoic acid, FAD. That was obviously new for today. But other than that, everything else was chemistry that you've already seen. And this really points out the point that I made earlier, that metabolism is really variations on relatively few reactions. We've just repurposed some of the same tricks, if you will, that we're used in glycolysis, and allowed it to now do an entire different pathway, the TCA cycle. It also points out how Hans Krebs was-- well, is still-remarkable, able to figure out from chemistry alone, because there's actually quite a bit of logic to the way metabolism works. Now, I want to say this again. Note there were two carbons that entered acetyl-CoA, and two carbons that were lost to CO2. But if you look, the green carbons remain in the same places until they get to succinate. and so the two carbons that enter are not lost on the first turn of the cycle. It's actually two carbons that came from oxaloacetate that are converted to CO2 as that acetyl-CoA goes through the cycle. And so to oxidize the exact carbons from acetyl-CoA to CO2 requires more than one turn of the cycle.

~34:07You'll also notice that the cycle is oxidation. And so oxidation reactions, of course, release energy. We've talked about that. And so it's favorable. And the products, if you will, are three NADH molecules. You can say plus one more NADH if we're going all the way from glucose or from pyruvate. Glucose derived pyruvate because the pyruvate dehydrogenase reaction also generates an NADH to make that a acetyl-CoA. One FADH2, as well as one GTP molecule. And so lots of oxidation going on here. We completely oxidized two carbons to CO2, so that's energy release. But you notice you only get one GTP from the molecule.

~34:49Now, this GTP, of course-- that reaction, the succinic thiokinase reaction, like the reactions we saw on glycolysis, is such that it can generate GTP at a high DGP-GDP ratio, or ATP-ADP ratio. Remember, those are equivalent. Those energy charge are similar, and so that makes sense. But most of the energy released is actually reducing NAD and FAD to NADH and FADH2. And of course, these need to transfer their electrons somewhere else, and that's the role of oxygen. Oxygen, remember, is a very good electron donor, and so it's the ultimate transfer of those electrons from these molecules to oxygen that also provide energy that the cell can use to do work, but it does so, in a way, by charging up different ratios in the cell. So the NAD-NADH or the FADH2-FAD ratios. And just like we talked about, the ratio or the energy of ATP is in the interconversion between ATP and ADP. It's the ratio that drives the free energy change. The same thing exists for an NADH and NAD, FADH2 and FAD. And so charging up these ratios while passing through the TCA cycle, and the ultimate downstream transfer of those electrons to oxygen, really is where most of the energy is captured as carbon is oxidized through the TCA cycle. And exactly how that works and how it can be related to ATP will be something that'll be more explicit in the coming lectures. Now, I want to point out apart from the oxidation, there's actually lots of intermediates made here. And it turns out, a bunch of these intermediates are useful for cells to make stuff. So we talked about gluconeogenesis. Gluconeogenesis needs electron balance. We get NADH from the TCA cycle, and so you can think of gluconeogenesis as an alternative to fermentation to dispose of electrons. Well, you can use the NADH from the TCA cycle to run gluconeogenesis as well. But beyond the cofactors, the carbon itself. So citrate, I've alluded to now a few times, is important as a precursor to make fat. We'll discuss that in later lectures, too. But other intermediates in this pathway are useful for various amino acids and nucleic acids. And so there's lots of things that can come from the TCA cycle that cells can find useful to do, not just catabolism, but also anabolic processes. Now, the way the TCA cycle works, though, is that there's actually an issue if you want to use the intermediates from the TCA cycle to make stuff. And so what is that issue? Well, the TCA cycle functions at a site as a cycle. And so, if we're going to take things in and out of it, that has consequences for how the cycle runs. Now there's a couple words for this that I want to just introduce to you. The first one is cataplerosis and the second one is anaplerosis. And so cataplerosis is the act of removing stuff from a metabolic cycle. So, we're going to remove citrate from the cycle to make fat. That's cataplerosis. And anaplerosis is adding stuff back to a metabolic cycle so it can continue to function. Viewing this is really evident if you think of the TCA cycle as a chicken and egg problem. So, the very first time acetyl-CoA was generated, how do you start the TCA cycle in the first place? You can't add it to the TCA cycle unless you have oxaloacetate to combine with the acetyl-CoA, which can then generate another oxaloacetate. So where does the first oxaloacetate come from? Well, we already talked about one reaction. We talked about it in the context of gluconeogenesis. That can solve this problem.

~38:47And so we have pyruvate. And so we talked earlier today how we can do oxidative decarboxylation of pyruvate to give acetyl-CoA. But we talked to in the gluconeogenesis lecture how we can add a CO2 pyruvate to generate oxaloacetate. So if I do those two reactions, now I have all the carbon I need to generate a citrate and start off the TCA cycle. Now obviously, if I do cataplerosis and I remove that citrate I made to make fat, well, now I need two pyruvate again to generate the next citrate if I'm using this pathway, because every time I bring an acetyl-CoA into the cycle, I need an oxaloacetate to combine it with. And so if I remove something, I have to add something back. And so pyruvate to oxaloacetate, the pyruvate carboxylase reaction is an example of an anaplerotic reaction. Now what this means, though, is that in order to do an anaplerosis, you have to be able to generate four-carbon oxaloacetate, or a four-carbon molecule. Now, pyruvate carboxylase, pyruvate to oxaloacetate, allows you to do that. We can take a three-carbon molecule and generate four-carbon oxaloacetate. However, if we start from a two-carbon molecule like acetate or acetyl-CoA that enters the cycle, it's actually not so simple to take that two-carbon molecule and turn it into four-carbon oxaloacetate. And in fact, humans lack any enzymes that allow them to take a two-carbon unit, to take acetate or acetyl-CoA, and turn it into anything that's longer than net-- turn it into anything that's longer than two carbons. And this has important implications for human physiology, because what it says is that we can't make glucose from anything that starts with something less than three carbons long. So if you drink alcohol and you metabolize that alcohol to acetate, it turns out you take fat and you break down fat also to acetyl-CoA, to acetate two carbons long. There is no way to turn those molecules into glucose, because you cannot generate the oxaloacetate to do the anaplerosis that's necessary to get it there. What this means is that our body can only store calories that come from two carbon units, fat or alcohol, as fat. We can never turn them back into glucose or make glycogen. And this is very relevant for those of you that go to medical school, because it's relevant to our physiology. And that is, when our bodies exhaust all of our stores of glucose, what happens? Our liver can no longer do gluconeogenesis. And so what happens? Now it has to switch over to doing something else. It has to work with two carbon units. And ultimately, this is ketone metabolism, which we'll talk about in a few lectures. It also said that the body-- that this is also the basis of a very old adage that's out there that some of you may have heard-- that you need to have some other fuel if you're going to burn fat. The basis for that is that fat is turned into two carbon units, acetyl-CoA units. And so if you're going to take those acetyl-CoA units and ultimately burn them away, turn them into CO2, you need a source of oxaloacetate, or your TCA cycle won't work. And you don't need a lot of something, but it is true. You can't start just with acetyl-CoA as a human and turn it into CO2, so you need some-- at least a little bit of oxaloacetate to get your TCA cycle started. Now, that's a problem that we as humans and other mammals face, but it turns out there's lots of microbes out there that grow just fine on acetate or on alcohol, even if it's only carbon source. And so those organisms must have some way to build stuff from two carbon units. That is a way to use two carbon units and do an anaplerosis. And it turns out the way they do this is via something called the glyoxylate cycle.

~43:06And so the glyoxylate cycle is an alternative version of the TCA cycle that effectively uses two enzymes that we lack as mammals. And so I'll quickly tell you about it here. So, this is isocitrate from the TCA cycle. And some microbes have an enzyme called isocitrate lyase. And what isocitrate lyase does is basically splits citrate in half, such that the top portion of the molecule is another TCA cycle intermediate, succinate. And the bottom portion of a molecule is this two carbon aldehyde called glyoxylate. Glyoxylate can react with acetyl-CoA by another enzyme that we lack as humans called malate synthase. And I don't have time to show the mechanism again, but malate synthase basically adds the two carbons from acetyl-CoA to the aldehyde, the carbonyl, the aldehyde carbonyl of glyoxylate in a reaction that is, for all intents and purposes, exactly what happens in citrate synthase. That will generate this molecule, which is malate, also in the TCA cycle. And so having these two extra reactions, isocitrate lyase and malate synthase, gives microbes the ability to have acetyl-CoA be anaplerotic. And so how does that work? Well, that's because if we start with one oxaloacetate, four carbons, and acetyl-CoA, two carbons, can run the TCA cycle and make citrate, six carbons. Turn that citrate into isocitrate. Use isocitrate lyase to generate glyoxylate, two carbons. Plus succinate. That succinate can run through succinate dehydrogenase to generate malate, which you can go through malate dehydrogenase to generate oxaloacetate. This glyoxylate can start with a second acetyl-CoA. Two carbons come together, generate malate. That generates a second malate molecule, which can then exit the cycle as malate or oxaloacetate or whatever you want. And so basically, it allows two acetyl-CoAs to net generate an oxaloacetate, and so net generates a way to do an anaplerosis from two carbon units by having this malate synthase reaction and this isocitrate lyase reaction and run this alternative version of the TCA cycle, called the glyoxylate cycle. And it's a nice way how life-- again, no new chemistry here, just variations on what we've already shown-- repurposed the similar chemistries that it's already using as a way to live off of carbon sources that contain only two carbons, like ethanol or acetate. So in closing today, the last thing I want to talk about, very briefly, is how the TCA cycle is regulated. And we don't need to spend a ton of time on this, because it really follows principles that make sense, particularly when we think about things that we've already talk about. And so, regulation of the TCA cycles is of course important, and it's really a critical hub, both for anabolic and catabolic pathways. So, you needed to get energy to fully oxidize carbon, but it's also a useful place to get stuff. And before I talk about regulation, I just want to point out that many of the enzymes in the TCA cycle, even though we talk about it in the mitochondria and we're about to talk about regulation in terms of catabolism-- that is oxidation, ways to release energy-- many of these enzymes are also in other locations in the cell, because there's functions for them in making stuff that is very different than what goes on in the TCA cycle. And the regulation that I'll tell you about, and that comes up on MCAT exams and stuff like that, usually talks about this pathway as a catabolic pathway, as a way to make CO2, to make ATP. However, recognize that there's also variations on this pathway that can use in anabolic things, making stuff. And that regulation is something that really is not necessarily what we're going to talk about here and is a little bit less well-understood.

~47:10But at least the regulation, in terms of catabolism, if we put it in context of glucose metabolism-- so here's glycolysis, turning glucose into pyruvate. And then that pyruvate can operate through the pyruvate dehydrogenase reaction to generate acetyl-CoA. That acetyl-CoA can combine with oxaloacetate to generate citrate. That citrate, of course, can be used to generate fat, as we've talked about. That can go to isocitrate. Isocitrate to alpha ketoglutarate. That's catalyzed by isocitrate dehydrogenase, which I will abbreviate IDH. Alpha ketoglutarate to succinate. More correctly, to succinyl-CoA by alpha ketoglutarate dehydrogenase, Alpha KGDH. And then this back to oxaloacetate. And so the main enzymes that are typically discussed as being regulated are alpha ketoglutarate dehydrogenase, isocitrate dehydrogenase, and pyruvate dehydrogenase. I also drew glycolysis. And I'll just write over here, gluconeogenesis up here, because we can now see more fully how this plays in with citrate acting as a positive regulator with gluconeogenesis and an inhibitor of glycolysis. Again, we're full in citrate, stop running glycolysis, start running gluconeogenesis. Now, the other regulation. If you have a lot of acetyl-CoA, stop making it from pyruvate. acetyl-CoA is a negative regulator of PDH. This is carbon oxidation. Releases a lot of energy, generates a lot of ATP, generates a lot of NADH. If you have lots of those things, no reason to keep sending carbon into acetyl-CoA to go into the TCA cycle. Ultimately, we have to send those electrons somewhere. So if there's nowhere to put them, low oxygen also inhibits pyruvate dehydrogenase. And of course, if you need more energy, if ADP is high, that activates pyruvate dehydrogenase. So at least in terms of glucose, complete glucose oxidation, a lot of regulation happens at pyruvate dehydrogenase. And a lot of it really makes sense. High levels of ATP, high levels of NADH, also inhibit isocitrate dehydrogenase and alpha ketoglutarate dehydrogenase. Makes sense. Succinol-CoA high, inhibit alpha ketoglutarate dehydrogenase. High levels of ADP, need to release more energy, activate alpha ketoglutarate dehydrogenase. Again, these are the feedbacks that people talk about on board exams. Good to know, but you can almost guess what they would be from first principles. Because remember, this is a pathway that releases a lot of energy. High energy high ATP, high NADH-- don't run the cycle, don't enter carbon in the cycle. Low energy, high ADP, put carbon in the cycle, run the cycle faster. Makes sense. Also makes sense of the reciprocal regulation of glycolysis and gluconeogenesis. Great. Next time, we will talk more about how we can oxidize fatty acids, fat, by accessing acetyl-CoA and entering it into the cycle. Thanks.

3.6TCA 循环的两用性、回补反应与乙醛酸循环：为什么植物能把脂肪变成糖而动物绝不能？

一、柠檬酸循环的两用性（Amphibolic Nature）：三大物质代谢的共同枢纽

如果仅仅把柠檬酸循环看作一台“把乙酰-CoA 烧成 CO₂ 的产能锅炉”，那就太小看它在细胞代谢网络中的崇高地位了。Vander Heiden 教授在 L23 后半段反复强调：两用性代谢途径（Amphibolic Pathway）：是指像柠檬酸循环这样，既在分解代谢（Catabolism）中充当糖、脂质和蛋白质彻底氧化供能的最终共同通路，又在合成代谢（Anabolism）中为葡萄糖、脂肪酸、胆固醇、氨基酸、卟啉（血红素）和核苷酸的生物合成提供关键碳骨架前体的代谢途径 2011/2012·简答。

让我们盘点一下，当细胞需要构建自身生物大分子时，是如何从 TCA 循环这口“大蓄水池”中向外抽调中间产物的——Vander Heiden 教授引入了一个专门术语：抽提反应（Cataplerosis，即从代谢循环中抽走中间物用于生物合成）：

1. 柠檬酸（Citrate） → 脂肪酸与胆固醇合成：当线粒体能量充沛、柠檬酸积累时，柠檬酸经三羧酸载体跨膜进入细胞质，被 ATP-柠檬酸裂解酶（ACL）裂解为胞质乙酰-CoA 和草酰乙酸，为脂肪酸和胆固醇的从头合成提供二碳原料。
2. α-酮戊二酸（α-KG） → 谷氨酸族氨基酸与嘌呤合成：α-KG 经转氨基作用或 L-谷氨酸脱氢酶还原氨基化生成谷氨酸（Glu），进而衍生出谷氨酰胺（Gln）、脯氨酸（Pro）、精氨酸（Arg），并为嘌呤和嘧啶核苷酸合成提供氮源。
3. 琥珀酰-CoA（Succinyl-CoA） → 卟啉与血红素合成：琥珀酰-CoA 与甘氨酸在 δ-氨基-γ-酮戊酸合酶（ALA 合酶，需 PLP）催化下缩合，是动物合成卟啉环、血红素（Heme，血红蛋白与细胞色素辅基）以及植物合成叶绿素的直接前体！
4. 草酰乙酸（OAA） → 天冬氨酸族氨基酸、嘧啶与糖异生：OAA 经谷草转氨酶（AST/GOT）转氨生成天冬氨酸（Asp），进而合成天冬酰胺（Asn）、甲硫氨酸（Met）、苏氨酸（Thr）、异亮氨酸（Ile）、赖氨酸（Lys）以及嘧啶环；或者经 PEPCK 转化为 PEP 进入糖异生合成葡萄糖！

二、回补反应（Anaplerotic Reactions）与“脂肪在糖的火焰中燃烧”

看到这里，一个严峻的逻辑矛盾浮出水面了：既然草酰乙酸（OAA）在 TCA 循环中扮演着“催化剂式四碳载体”（1 分子 OAA 接纳 1 分子乙酰-CoA，转一圈回来还是 1 分子 OAA），那么一旦草酰乙酸、柠檬酸或 α-酮戊二酸被抽走去合成氨基酸、脂肪酸或葡萄糖（Cataplerosis），循环里的四碳载体岂不是立刻亏空枯竭，导致整个 TCA 循环彻底停摆吗？

为了防止蓄水池干涸，细胞进化出了一套至关重要的补水机制——回补反应（Anaplerotic Reaction，又称填充反应）：是指当柠檬酸循环的中间产物被抽调用于其他生物合成途径而减少时，由循环以外的酶促反应生成四碳或五碳中间产物（主要是草酰乙酸）予以补充，以确保柠檬酸循环得以持续正常运转的反应 2008·名解2022·简答。

三、乙醛酸循环（Glyoxylate Cycle）：油料种子的“化脂为糖”魔法

既然人类和哺乳动物无法把二碳的乙酰-CoA（来自脂肪或酒精）净转化为四碳的草酰乙酸或六碳的葡萄糖，那么大自然里有没有生物能打破这个魔咒呢？

想一想：一颗埋在黑暗泥土里正在萌发的向日葵、花生或蓖麻种子（油料作物种子），在长出绿叶进行光合作用之前，它体内储存的几乎全都是脂肪（三酰甘油），却急需大量葡萄糖和纤维素来建造细胞壁、供给胚芽生长！再想一想，像大肠杆菌（E. coli）或酵母这样的微生物，哪怕你只在培养皿里给它喂二碳的乙酸（醋酸盐）或乙醇作为唯一碳源，它照样能合成出所有的氨基酸、核苷酸和葡萄糖，活得生龙活虎！它们是怎么做到的？

答案是：植物（特别是油料种子）和部分微生物（细菌、真菌、藻类）拥有一条动物在进化中彻底丢失的“TCA 循环捷径”——乙醛酸循环（Glyoxylate Cycle，又称乙醛酸途径）：是存在于植物（油料种子萌发期的乙醛酸循环体）和某些微生物中的三羧酸循环支路，通过异柠檬酸裂解酶和苹果酸合酶两个特有酶，绕过 TCA 循环的两次氧化脱羧步骤，将 2 分子乙酰-CoA（2C）净合成为 1 分子四碳二羧酸（琥珀酸/苹果酸/草酰乙酸，4C），进而通过糖异生途径将脂肪（或乙酸）转化为糖类的代谢过程 2012/2013/2015/2019/2025·名解！

1. 乙醛酸循环的五步反应与“绕开两次脱羧”的核心逻辑

在植物油料种子细胞中，乙醛酸循环发生在一种特化的过氧化物酶体——乙醛酸循环体（Glyoxysome）与线粒体、细胞质的紧密协作中 2007/2009/2010·单选2025·填空：

1. 第 1 步（同 TCA）：在乙醛酸循环体内，第 1 分子乙酰-CoA（2C，来自脂肪酸 β-氧化）与草酰乙酸（4C）在柠檬酸合酶催化下缩合为柠檬酸（6C）；
2. 第 2 步（同 TCA）：柠檬酸经顺乌头酸酶异构为异柠檬酸（6C）；
3. 第 3 步（分叉点 · 特有酶 1：异柠檬酸裂解酶 Isocitrate Lyase, ICL）：
   异柠檬酸（6C） ──(异柠檬酸裂解酶（乙醛酸循环体特有）)──► 琥珀酸（Succinate，4C） + 乙醛酸（Glyoxylate，OHC-COO^-，2C）

• 化学本质：这是一步逆羟醛缩合（retro-aldol cleavage）反应！异柠檬酸裂解酶完全不进行氧化脱羧（不丢掉一分一毫的碳原子！），而是像一把剪刀把六碳异柠檬酸直接裂解为一分子四碳的琥珀酸（4C）和一分子二碳的乙醛酸（Glyoxylate，2C）！

1. 第 4 步（特有酶 2：苹果酸合酶 Malate Synthase, MS）：
   乙醛酸（2C） + 乙酰-CoA（第 2 分子，2C） + H_2O ──(苹果酸合酶（乙醛酸循环体特有）)──► L-苹果酸（Malate，4C） + CoA-SH + H⁺

• 化学本质：正如 Vander Heiden 教授所指出的，苹果酸合酶的机理与柠檬酸合酶完全一样——利用第 2 分子乙酰-CoA 的高能硫酯键水解供能，将其二碳乙酰基缩合到二碳乙醛酸的醛羰基上，直接生成四碳的 L-苹果酸（4C）！
• 高频易混陷阱：乙醛酸循环的第二个关键酶叫苹果酸合酶（Malate synthase，催化 2C 乙醛酸 + 2C 乙酰-CoA → 4C 苹果酸），千万不要写成苹果酸脱氢酶（催化苹果酸 ↔ 草酰乙酸）或苹果酸酶（催化丙酮酸 + CO₂ ↔ 苹果酸）！

1. 第 5 步（载体再生与净产物输出糖异生）：

• 乙醛酸循环体内生成的 L-苹果酸被苹果酸脱氢酶氧化再生为草酰乙酸（4C）（产 1 NADH），继续留在循环体内接纳下一轮的乙酰-CoA；
• 而第 3 步切下来的那分子净赚的琥珀酸（4C）则跨膜转运进入线粒体，借用线粒体 TCA 循环后半段的酶系（琥珀酸脱氢酶产 1 FADH₂ → 延胡索酸酶 → 苹果酸脱氢酶产 1 NADH）转化为苹果酸 / 草酰乙酸，随后进入细胞质经 PEPCK 和糖异生途径源源不断地合成为葡萄糖和蔗糖 2025·填空！

把乙醛酸循环与线粒体琥珀酸氧化合起来，每投入 2 分子乙酰-CoA 的总反应式为：

2 乙酰-CoA（2C× 2） + 2 NAD⁺ + FAD + 2 H_2O ⟶ 草酰乙酸（4C，净生成！） + 2 CoA-SH + 2 NADH + FADH₂ + 2 H⁺

2. 异柠檬酸分叉点的共价修饰开关：细菌如何决定走 TCA 还是走乙醛酸循环？

在大肠杆菌等微生物中，TCA 循环和乙醛酸循环都在细胞质中进行，共用异柠檬酸这同一个分叉底物。细胞如何决定把异柠檬酸送给异柠檬酸脱氢酶（IDH，走 TCA 烧掉产 ATP），还是送给异柠檬酸裂解酶（ICL，走乙醛酸循环攒碳造糖）？

• 当细菌能量匮乏（ADP、AMP 高，中间产物低）时：双功能激酶/磷酸酶发挥磷酸酶活性，将异柠檬酸脱氢酶（IDH）去磷酸化而激活（同时 AMP 等别构抑制异柠檬酸裂解酶），由于 IDH 对异柠檬酸的亲和力比裂解酶高得多，异柠檬酸优先流入 TCA 循环彻底氧化供能；反之，当能量充沛时，IDH 被磷酸化而失活，异柠檬酸分流进入乙醛酸循环合成四碳骨架和糖类！
• 当细菌生长在乙酸培养基上且能量充沛（ATP、NADH、3-PG、PEP 等丰富）时：双功能酶发挥蛋白激酶活性，将 IDH 磷酸化而失活，同时高浓度中间产物激活异柠檬酸裂解酶（ICL），关上脱羧大门，让异柠檬酸全速涌入乙醛酸循环合成四碳骨架和葡萄糖！

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🎬 MIT 7.05 Lec 23 · TCA Cycle II, Isotope Tracing & Glyoxylate Cycle⏱ ~50:00

~00:00MITOCW | 23. TCA Cycle II

~00:02MATTHEW

~00:02Hello, everybody. Last time, I introduced the idea of the TCA cycle, tricarboxylic acid cycle. Also known as the

~00:10VANDER

~00:10citric acid cycle, because citric acid is a tricarboxylic acid, as you'll see later today. Also known as the Krebs

~00:18HEIDEN:

~00:18cycle, named after Hans Krebs, who discovered it in the early part of the last century. The TCA cycle is the series of reactions that occurs in the mitochondrial matrix and it allows the complete oxidation of two carbon units, derived from many things, including pyruvate, derived from glucose and glycolysis and enables the complete oxidation of that carbon to CO2. Now, it's a cycle because those two carbon units from pyruvate or other sources enter the cycle, combine with 4carbon oxaloacetate, and combine to make 6-carbon citrate. Hence the citric acid cycle, or TCA cycle, that is then oxidized back to 4 carbon units, forming a cycle that allows cells to release lots of energy. Complete oxygen enables, ultimately, complete oxidation of glucose to CO2. We've talked many times how this releases energy. And it also generates lots of intermediates for the cell that can be used to make other stuff. Last time, I alluded to the fact that citrate can be used, say, to make fatty acids. Today, what I want to do is I want to dive into the details of how this TCA cycle occurs and the consequences of the way it works, and how that affects other aspects of metabolism. You'll see that it actually affects the ability of different organisms, whether or not they can make things from intermediates in this cycle, because of how the cycle works. Now, to start. Of course, if we're going to start from pyruvate. Pyruvate, of course, has three carbons. And so if we're going to generate a two-carbon acetate group, we have to lose CO2. And as I described a couple lectures ago, acetate is also the thing that we can derive from metabolism of ethanol, showing us two things-- metabolized glucose to pyruvate or ethanol itself-- can both be turned into acetate. But really, the donor is this molecule, acetyl-CoA. Which is basically also a carboxylic acid, but rather than being a carboxylic acid, instead makes this a thioester bond, which activates this carbon as a leaving group, such that it can combine with oxaloacetate, which you'll remember from gluconeogenesis, releasing that S-CoA molecule to generate six-carbon citric. So, this is citric. You can see that we've made a bond from this carbon on the acetate, losing this S-CoA group to this carbon oxaloacetate to make this six-carbon citrate molecule, which is a one, two, three-- three-carboxylic acid, or tricarboxylic acid. Hence the name citric acid cycle, tricarboxylic acid cycle.

~02:58Now, these six carbons can then-- or this six-carbon citrate molecule can then be oxidized, generating two CO2 molecules that are released, and ultimately reforming oxaloacetate that can pick up another two-carbon acetylCoA to generate another citrate, and around and around the cycle goes, allowing in the end the net entry of two carbons, effectively from acetate, and release of two carbons as CO2. Now, as I alluded to, this can come from pyruvate. It can come from acetate itself, vinegar. It can come from alcohol. It turns out that when you break down fat, you also break it into two carbon units. And so this cycle becomes very useful for cells, because it allows the oxidation of many different molecules to completely turn that carbon into CO2. Now, if we're going to do this from glucose, however, you'll remember that pyruvate has three carbons. And so if we're going to turn pyruvate into acetate, or acetyl-CoA, we have to lose a carbon of CO2. We have to lose this carbon as CO2. Now, we saw this before, that we can do this via-- this is exactly how we generated ethanol when we did fermentation of pyruvate to ethanol, but that molecule didn't generate acetate, It generated acetaldehyde. The difference, of course, being whether or not this carbon gets oxidized in the case of acetate to the acid, or in acetaldehyde in ethanol metabolism, where it remains an aldehyde. Now obviously, you can make ethanol and then turn that ethanol into acetate. And that's a pathway that I guess certainly would work. However, that's not the way it happens in most organisms. Most organisms actually directly produce acetate, or more correctly, acetyl-CoA from pyruvate. So let's take a look at that reaction. So here once again is pyruvate. And if we turn that pyruvate into this two-carbon acetyl-CoA, let's look here what has to happen. Well, the first thing is that we have to decarboxylate this molecule. And so that generates a CO2. We have to lose that one carbon. Also, as I pointed out, if we do this as if we did it in ethanol metabolism, we'd be left with an aldehyde. But this is an acid, and so this carbon also has to be oxidized. We know how to do that. We can donate those electrons to something like NAD, so the NAD is reduced to NADH. And we had to add this S-CoA molecule, which I'll come to in a minute. And so each of these steps ends up making a fairly complicated reaction. Obviously, several co-factors are going to be involved. You should be able to guess that just by looking at it. Obviously, I already drew up there NAD. If we're going to decarboxylate, remember this is a alpha carboxylic, an alpha ketoacid-- the ketone group is alpha to the carboxylic acid. And so if we're doing alpha decarboxylation, as you might guess, we need a cofactor. That co-factor, as I told you before, is the co-factor factor thiamine pyrophosphate. And finally, there's this S-CoA group. We need to describe what that is. However, before delving into those, and there's actually a couple other factors that are needed as well, I want to mention one other issue about this reaction, in that this reaction happens in the mitochondria, because that's also where the TCA cycle happens. So, recall that if we divide the cell into two compartments like an eukaryotic cell, here we have the cytosol and the mitochondria in a eukaryotic cell. As we've already talked about, having different compartments helps facilitate different metabolic reactions, because you can have different conditions in the two compartments.

~06:54And so as we said, glycolysis occurs in the cytosol. Glucose to pyruvate. And last time I mentioned, the TCA cycle occurs in the mitochondria. And so that means if we're going to fully oxidize the pyruvate carbon to CO2 using the TCA cycle in the mitochondria, that pyruvate has to get from the cytosol to the mitochondria, or at least carbons from the pyruvate have to get there. It turns out, you'll see in a minute, acetyl-CoA is a very large group. And so pyruvate itself is transported into the mitochondria. And that's where the reaction occurs to turn it into acetyl-CoA that can then enter the TCA cycle and oxidize. However, what this means is that a transporter is actually needed to get this across the mitochondrial membranes and into the matrix of the mitochondria where the TCA cycle happens. And I like to mention this because it turns out the pyruvate carrier-- that is, the transporter-- the way that it actually gets that pyruvate from the cytosol into the mitochondria actually was an unknown thing about metabolism until 2012, so not that long ago. Sometimes one can be left with the feeling, listening to these metabolism classes or reading a textbook, that everything about metabolism has been known forever, but it's actually not true. Here's a very key, central part of the pathway that was actually just discovered, at least at the time of this lecture, less than 10 years ago. It also illustrates that not all metabolism is completely understood. Now, let's get back to this reaction, how you turn pyruvate into acetyl-CoA. And I want to go through now and point out that several co-factors are needed. And so I already mentioned one of them, S-CoA, which is shorthand for coenzyme A. So, I need to tell you what that is. You hopefully are already familiar with TPP plus, thiamine pyrophosphate. We talked about that when we talked about how you do alpha decarboxylation to generate ethanol from pyruvate, also used here for the alpha decarboxylation reaction. Redox reaction happens, and so we needed NAD plus to get converted into NADH. We talked a lot about how that serves as an electron carrier, but it turns out that there's two additional electron carriers that are involved in this reaction. One of them is called FAD and the other one is called lipoic acid. Now you might say, why do we need all these electron carriers? Well, these are just different molecules that can carry two electrons, similar to NADH. And effectively, what these can do by having multiple electron carriers-- one can build chains of oxidation and reduction reactions. And it turns out these chains of oxidation reduction reactions really become central to energy transfers in biology, because building these chains allows more easy stepwise release of energy as one moves across these oxidation reduction reactions. Which remember, as I alluded to earlier, really are at the core of bioenergetics and a lot of what allows energy release from these pathways. What I mean by this will be more explicit as we go through what some of these co-factors look like. So, I'm not going to draw TPP plus or NAD again, but let's define what some of these other cofactors look like. So, let's start with coenzyme A. So, coenzyme A, it turns out, is useful. It's actually involved in lots of acylation reactions.

~10:34What's an acylation reaction? Well, that's basically if you're making a carbon-carbon bond by adding a molecule of greater than one carbon, so two carbons or greater, to something else. That's an acylation reaction, as we did with adding the two-carbon acetate to oxaloacetate to make citrate. And you'll actually see coenzyme A will come up in this in many, many lectures throughout the rest of the course.

~11:00Why this becomes useful is because it activates this basically carbon to the left of, in this case, the ketone. Because, by having that thioester there, ends up activating that carbon and allows it to carry out these acylation reactions. Coenzyme A itself is derived from a vitamin called pantothenic acid. And as a vitamin, this is something that you have to get from the diet. Now, when we abbreviate coenzyme A, which is often abbreviated S-CoA, you get the sense that it's this little tiny molecule that's just stuck on sulfur-- stuck on to the end to make this thioester bond. But it turns out coenzyme A is actually a giant molecule, one of the reasons why you actually synthesize acetyl-CoA in the mitochondria, because it's hard to transport this giant molecule. And so this is what pantothenic acid looks like. So, if I put an acid group here and a hydroxyl group there, that would be pantothenic acid, the thing that's in your cereal, on the side of your cereal box. Then there's these additional pieces. This is the active end of the molecule, that's that sulfur from the S-CoA, and then this side of the molecule is esterified to two phosphates, which are esterified to-- I'm not going to draw it out, but this would have an adenine base and a phosphate there. So basically, this is ADP, with a phosphate added to the 3 prime position of the ADP molecule, added to pantothenic acid, added to this short chain with the sulfur on the end. And this whole molecule together is coenzyme A. And so when we say acetyl-CoA, it's this giant molecule, S-thioester to the carbonyl, the acid on acetate, to CH3. And so that would be, basically, acetyl-CoA. And so one convenient thing about this is it's much easier than drawing that big molecule, but it is a little bit misleading in terms of its size. So, that's coenzyme A. Next one I want to talk about is FAD which stands for flavin adenine dinucleotide. So, flavin adenine dinucleotide is an electron carrier, just like NAD plus. And it's derived from the vitamin riboflavin, also referred to as vitamin B2. Another thing from the side of your cereal box, and basically looks like this. So, like NAD, it's a dinucleotide. And so here's ADP, just like we do for CoA, or just like one end of NAD. One difference is that the other nucleotide down here actually isn't technically a sugar. It's ribitol instead of ribose. What does that mean? It doesn't have an aldehyde. Instead, it is just a five-carbon chain where all of the carbons are alcohols. And so since there's no aldehyde, it doesn't form a ring. And the base on this end, as a nicotinamide, is this flavin group, which looks like this. And so this is FAD, which is in the oxidized form. Turns out, in the oxidized form, FAD is yellow, hence riboflavin-"flavin", yellow. And the reason it's yellow-- you see there's a conjugated double bond here across this part. It turns out this is the active part of the molecule, and it works as follows. And so if you have a hydride ion, remember the way we can transfer two electrons. Can transfer the two electrons that way, and that allows it to generate. And I'll just draw the middle, here. Active part of the molecule. So, that would be these two nitrogens. We've added two electrons to it. And so this is abbreviated FADH2, or the reduced form of FAD. And it is colorless, because now you no longer have that conjugated double bond system, loses its color. And so you can follow whether FAD is oxidized or reduced by just looking at color change. So, that's FAD. It's an electron carrier, carries electrons by a hydride transfer, very similarly to what we described for NAD, but obviously a different molecule.

~15:13And the last molecule, the last co-factor that we need for this reaction, is lipoic acid. Which, unlike most things in metabolism, doesn't have an abbreviation and also functions as an electron carrier. And so lipoic acid looks like this. So, that is lipoic acid. This is in the oxidized form. And so where it's oxidized is here at this sulfur-sulfur bond. So, you can think of this as the same as a disulfide bond that you learned about from Professor Yaffe in proteins. And so this is the oxidized form of the disulfide bond. If I take this hydride, transfer two electrons across that bond, then it goes to-- and I'll just draw here the active end. Then we go here to the reduced form of lipoic acid, and there's no abbreviation for oxidized or reduced lipoic acid. It's just lipoic acid, oxidized lipoic acid, reduced. And how they're oxidized and reduced is basically very similar to the disulfide bonds that you saw in proteins being oxidized, disulfide bond being oxidized, or it can be reduced to not be a disulfide bond. Now, it turns out that these cofactors, our TPP, FAD, and lipoic acid, are all stably associated with different subunits of a multi-protein complex that assembles to catalyze that reaction to convert pyruvate to acetyl-CoA. The enzyme or enzyme complex that carries this out is referred to as PDH, sometimes abbreviated PDC, which stands for the pyruvate dehydrogenase complex. So, pyruvate dehydrogenase, PDH, pyruvate dehydrogenase complex, PDC. This complex is basically three different polypeptides that come together to form a complex and catalyze that reaction. Now, where this complex sits-- so, this is the mitochondria. This is the matrix. That's where this pyruvate dehydrogenase reaction occurs. That's where the TCA cycle occurs. And this PDH complex is basically sitting here at the membrane on the matrix side of the membrane. Now, the three different polypeptides that come together to form this complex are creatively named E1, E2, and E3, four. Enzyme one, two, and three. And each of these, as I alluded to, is associated with a different co-factor. And so E1 is associated with thymine pyrophosphate. E2 is associated with lipoic acid. And E3 is associated with FAD. Now, let's go through the mechanism for how this pyruvate dehydrogenase reaction works. So remember, this is the active part of TBP plus. It is bound in the active site of E1. And it reacts with pyruvate to catalyze alpha decarboxylation, just like we described for conversion of pyruvate to alcohol. So, you're going to see exactly the same mechanism that we drew before. So, that decarboxylates the alpha-keto acid, just like we saw to generate acetaldehyde. The difference is rather than resolve this such that this carbon has the same oxidation state and make acetaldehyde, instead the next step of this reaction is going to be oxidized by reducing lipoic acid. So, the active part of lipoic acid that is in the active site of the E2 subunit. So this will now regenerate E1, but what we're left with, then, is this. Now, this intermediate bound to E2. Here's where coenzyme A can come in, which will then generate acetyl-CoA. But now we are left with E2 in the reduced state, rather than being in the oxidized state. So E2 has to be re-oxidized in order for this complex to carry out the next catalytic cycle, and the way that works is as follows.

~18:56So you have FAD bound on E3. And so you have a hydride ion from the oxidation of E3 that can be transferred to FAD. That will generate FAD from the oxidized to the reduced form. So re-oxidize lipoic acid on E2, reduce FAD to FADH2, and then that FADH2 can be re-oxidized back to FAD via transferring those electrons to NAD plus to generate NADH. So in this case, FADH2 re-oxidized the FAD, NAD plus reduced to NADH. And so, effectively, what this happens is that enzyme E1 and E2 cooperate to call what's referred to as oxidative alpha decarboxylation of pyruvate, while adding -CoA, so that's where you get acetyl-CoA, with reduction of lipoic acid in E2, and then E3 re-oxidizes the lipoic acid in E2 while generating NADH. This NADH, once it's generated, of course those electrons have to go somewhere, so they, like in glycolysis-- it also needs to regenerate to NAD at some point. This is ultimately the electrons that end up on oxygen, and it's really this series of electron transfers, with oxygen being a good electron acceptor, that ultimately allows controlled energy release during carbon oxidation. And cells, you'll see, can use that to make ATP or do other kinds of work. So, the net reaction and/or another way to draw the pyruvate dehydrogenase reaction would be as follows, and that's taking pyruvate to acetyl-CoA. And so we're going to take coenzyme A and release CO2. This is done by TPP plus, as part of E1. That involves converting the lipoic acid in E2 from the oxidized to the reduced state. That lipoic acid then has to be re-oxidized. If something's oxidized, something else has to be reduced. That's FAD on E3, which also then cycles between the oxidized and reduce state. And ultimately, those electrons ended up being transferred to NAD plus to generate NADH. And so the PDH complex is basically a chain of electron transfer reactions. And it's the first example of a chain of electron transfer reactions. We're going to see that there is the electron transport chain in the mitochondria, effectively does the same thing. And by coupling oxidation and reduction reactions across chains of molecules like this, effectively as a preview, allows the stepwise energy release of these oxidation reactions to occur. So remember, if we burn glucose, completely oxidize it in one step, where those electrons are directly transferred to oxygen in combustion. Lots of energy released, but all in one step. By doing these stepwise electron transfers, we can then basically break up that energy release in a way that can be captured by cells to do work. Here, the way energy is captured, it's not so obvious in this electron transport reaction. But effectively, you're using oxidation of the ketone on pyruvate, with decarboxylation to the acid to generate, rather than just the acid, a thioester bond. And that's that thioester bond-- as well as NADH, that's energy as well-- but it's really that thioester bond that then can be recaptured later to drive synthesis of citrate in the TCA cycle. Now, next what I want to do is I want to discuss the TCA cycle reactions. Now that you see how you can get acetyl-CoA, at least from pyruvate, I want to discuss how you can now use that acetyl-CoA and oxidize it back to combine it with oxaloacetate, and oxidize it back to make citrate, and then oxidize it back to oxaloacetate to run the TCA cycle. OK, great. So, the first reaction of the TCA cycle. Here's acetyl-CoA. It will combine with oxaloacetate. This reaction is catalyzed by an enzyme called citrate synthase, and generates the six-carbon tricarboxylic acid citrate. So, how does this reaction work?

~22:58Here's drawing acetyl-CoA in a slightly different way. If I redraw this as the enol, this will generate citrate via, effectively, that mechanism. Next, citrate is converted by adding water across this carbon-carbon, or by removing water across this carboncarbon bond, to generate this intermediate called cis-aconitate. So this intermediate is called cis-aconitate. so all I did was dehydrate across that bond so there's a double bond there. And then if I re-add water across that bond, I generate this molecule called isocitrate. So effectively, to convert citrate to isocitrate, I'm moving the hydroxyl group from that carbon to this carbon. To do that, I basically dehydrate, make a double bond, remove water, readd water across that bond in the opposite direction to generate this molecule, isocitrate. This reaction is carried out by an enzyme called aconitase, and converts citrate to isocitrate. I think it's a little easier to see this reaction if I draw it a slightly different way. So, this here is just drawing citrate by just slightly rotating the molecule to look like that. And so I'm basically removing water here. And then I'm now just adding water back in the opposite orientation to generate isocitrate. Now, you'll notice when I drew this-- if you look at citrate, this is actually a symmetrical molecule, so the top half and the bottom half of citrate are identical. And so what's interesting about nature is that it treats these carbons- the green carbons that came from acetyl-CoA-- different from the side of the molecule that comes from oxaloacetate. And effectively, nature always moves the hydroxyl group to this carbon that came from oxaloacetate, and never moves it to this carbon that came from acetyl-CoA. This is an example where enzymes-- nature treats a symmetrical molecule like citrate in an asymmetrical way. And this has consequences for how carbon is actually traced through the entire TCA cycle. Because even though you might think things could get scrambled at citrate, they never do. Meaning an isocitrate-- it's always these green carbons that came from acetyl-CoA. You never get those green carbons on the other side of isocitrate. And so when we go through the TCA cycle, I'll keep these carbons green until the point where you can no longer distinguish which carbon came from came from which reaction. The next reaction is we're going to oxidize this carbon of isocitrate. So if we're going to oxidize that carbon, those electrons have to go somewhere. And so if we oxidize the carbon, we can use NAD plus as an electron acceptor, reduce it to NADH. That generates this intermediate. So hopefully this is clear to everybody at this point, but just in case. So, this carbon here and isocitrate. If I oxidize that alcohol to the ketone, now I generate a hydride ion. Those two electrons and the hydrogen can go to NAD plus and reduce it to NADH. That generates this intermediate. It's called oxalosuccinate. Which then, if you notice, the oxalosuccinate is now a beta keto acid. So alpha, beta. The acid group is beta to the ketone, so it's a beta keto acid. Remember, beta decarboxylation is favorable, and so I can lose that CO2. And what I'm left with is this molecule, which is called alpha keto glutarate. So this whole reaction here, the oxidation of the alcohol to the ketone, followed by beta decarboxylation of oxalosuccinate to alpha ketoglutarate, is carried out by an enzyme called isocitrate dehydrogenase. And of course, just to remind you, here's that beta keto acid in oxalosuccinate. And so that can decarboxylate, leading this enol. Which can, of course, rearrange back to the ketone that we see, an alpha ketoglutarate.

~26:57Now, if you look at alpha ketoglutarate, you'll notice that-- and I will redraw over here. So, this here is just me redrawing alpha ketoglutarate, which is often abbreviated alpha kG. Just drew it as a straight line. Now, if you'll notice, alpha keto glutarate is a alpha ketoacid. And so here the acid group is alpha to the ketone. So, an alpha keto acid. It's effectively just like pyruvate, but has this additional pieces on it. And it turns out the next step in the TCA cycle is the exact reaction that we saw with pyruvate dehydrogenase. It's alpha decarboxylation, oxidative alpha keto acid decarboxylation, as follows. So, we carry out. This is a molecule called succinyl-CoA. And so, you'll see what happened there is decarboxylated here, this carbon, while oxidizing this ketone to the acid and adding -CoA. That's a redox reaction. So NAD, NADH, it turns out this is exactly the same mechanism-- so I don't need to draw it again-- that I just showed you for pyruvate dehydrogenase. So it needs all the same cofactors-- TPP plus, lipoic acid, FAD. And in fact, it even shares some of the same enzyme complexes, subunits, as pyruvate dehydrogenase. And so this is a reaction that's catalyzed by alpha ketoglutarate dehydrogenase. Like pyruvate dehydrogenase, this is a complex, and so it has a unique E1, which makes sense. Remember, E1 of pyruvate dehydrogenase was actually the subunit that bound the pyruvate. E1 of alpha ketoglutarate dehydrogenase is unique. It binds alpha ketoglutarate instead of pyruvate. However, they share the same E2 and E3s, and so the E2s and E3s would carry out exactly the same reaction and play the same part in the mechanism of how you do this alpha oxidative alpha decarboxylation to turn alpha ketoglutarate into succinyl-CoA. Now, remember in pyruvate dehydrogenase, once we got that acetyl-CoA, we then use this CoA group to drive condensation with citrate. Well, in this case, what happens is you don't want to combine condensation here. Instead, what is going to happen is you want to use this CoA group to now generate ATP. And so this is going to couple release of the CoA to generate an ATP equivalent. It's actually GTP that's generated by the TCA cycle. And so, this is going to generate this molecule, succinate. And the enzyme that does this is called succinic bio-kinase. So, let's go through over here how this enzyme works. So here's succinyl-CoA, basically using favorable loss of the CoA breaking the thioester bond to generate this acid anhydride. We saw an acid anhydride before in glycolysis. Remember, we made 1-3-bisphosphoglycerate, so that's a good phosphate donor. And then that can be used to generate succinate, and transferring that phosphate to GDP to make GTP, just like 1-3bisphosphoglycerate, was able to transfer the phosphate from the acid anhydride to ADP to make ATP in glycolysis. So, the next step is to oxidize this carbon-carbon bond in succinate. So if we're going to oxidize a carbon-carbon bond, those electrons have to go somewhere. So there's our carbon-carbon bond. And so if we generate hydride ion just like we did in other oxidation reactions, but this hydride ion is transferred not to NAD, but instead to FAD, a different electron carrier, to generate if FADH2. Now, I should point out succinate, like citrate, is a symmetrical molecule. But at this point, nature doesn't tell the difference. Once it generates succinate, this molecule now gets scrambled. And so everything downstream of succinate, you no longer know which carbons came from acetyl-CoA. This generates this molecule called fumarate, and this reaction is carried out by an enzyme called succinate dehydrogenase, often abbreviated SDH for succinate dehydrogenase.

~30:56Now, the next reaction is, we're going to add water across this double bond of fumarate. And that generates this intermediate, malate. This reaction is carried out by an enzyme called fumarate hydratase. And it's simply adding a water molecule across that double bond. Once we have malate, if you look what's the difference between malate and oxaloacetate, the difference is that in oxaloacetate, this carbon is a ketone. Whereas in malate, it's an alcohol. And so if we want to turn this carbon from the alcohol into the ketone, that is, of course, a oxidation reaction, and so those electrons have to go somewhere. Don't need to draw the mechanism again. It's basically just the hydride transfer to oxidize this to an alcohol, to the ketone. That oxidation couples to a reduction of NAD plus to NADH. This is carried out by an enzyme called malate dehydrogenase. And doing this completes the TCA cycle, regenerating oxaloacetate, that can then recombine with another acetyl-CoA to go through another round of the cycle. You'll notice that going through this cycle, there's two CO2s lost. One of them is lost here at the isocitrate to alpha ketoglutarate reaction. So, this decarboxylation from oxaloacetate to alpha ketoglutarate. That beta decarboxylation, that's the first CO2. The other one is lost here at the alpha ketoglutarate. The alpha ketoglutarate dehydrogenase step, where you have this alpha decarboxylation to take alpha ketoglutarate to succinyl-CoA oxidative alpha decarboxylation. Now, what's cool about this, as you noticed, we just discussed all the reactions of the TCA cycle. And I showed you, reminded you, of some chemistry that you've already seen, but unless you count the chemistry we showed you earlier for how the PDH and alpha ketoglutarate dehydrogenase reactions work with E1, E2 and E3, with lipoic acid, FAD. That was obviously new for today. But other than that, everything else was chemistry that you've already seen. And this really points out the point that I made earlier, that metabolism is really variations on relatively few reactions. We've just repurposed some of the same tricks, if you will, that we're used in glycolysis, and allowed it to now do an entire different pathway, the TCA cycle. It also points out how Hans Krebs was-- well, is still-remarkable, able to figure out from chemistry alone, because there's actually quite a bit of logic to the way metabolism works. Now, I want to say this again. Note there were two carbons that entered acetyl-CoA, and two carbons that were lost to CO2. But if you look, the green carbons remain in the same places until they get to succinate. and so the two carbons that enter are not lost on the first turn of the cycle. It's actually two carbons that came from oxaloacetate that are converted to CO2 as that acetyl-CoA goes through the cycle. And so to oxidize the exact carbons from acetyl-CoA to CO2 requires more than one turn of the cycle.

~34:07You'll also notice that the cycle is oxidation. And so oxidation reactions, of course, release energy. We've talked about that. And so it's favorable. And the products, if you will, are three NADH molecules. You can say plus one more NADH if we're going all the way from glucose or from pyruvate. Glucose derived pyruvate because the pyruvate dehydrogenase reaction also generates an NADH to make that a acetyl-CoA. One FADH2, as well as one GTP molecule. And so lots of oxidation going on here. We completely oxidized two carbons to CO2, so that's energy release. But you notice you only get one GTP from the molecule.

~34:49Now, this GTP, of course-- that reaction, the succinic thiokinase reaction, like the reactions we saw on glycolysis, is such that it can generate GTP at a high DGP-GDP ratio, or ATP-ADP ratio. Remember, those are equivalent. Those energy charge are similar, and so that makes sense. But most of the energy released is actually reducing NAD and FAD to NADH and FADH2. And of course, these need to transfer their electrons somewhere else, and that's the role of oxygen. Oxygen, remember, is a very good electron donor, and so it's the ultimate transfer of those electrons from these molecules to oxygen that also provide energy that the cell can use to do work, but it does so, in a way, by charging up different ratios in the cell. So the NAD-NADH or the FADH2-FAD ratios. And just like we talked about, the ratio or the energy of ATP is in the interconversion between ATP and ADP. It's the ratio that drives the free energy change. The same thing exists for an NADH and NAD, FADH2 and FAD. And so charging up these ratios while passing through the TCA cycle, and the ultimate downstream transfer of those electrons to oxygen, really is where most of the energy is captured as carbon is oxidized through the TCA cycle. And exactly how that works and how it can be related to ATP will be something that'll be more explicit in the coming lectures. Now, I want to point out apart from the oxidation, there's actually lots of intermediates made here. And it turns out, a bunch of these intermediates are useful for cells to make stuff. So we talked about gluconeogenesis. Gluconeogenesis needs electron balance. We get NADH from the TCA cycle, and so you can think of gluconeogenesis as an alternative to fermentation to dispose of electrons. Well, you can use the NADH from the TCA cycle to run gluconeogenesis as well. But beyond the cofactors, the carbon itself. So citrate, I've alluded to now a few times, is important as a precursor to make fat. We'll discuss that in later lectures, too. But other intermediates in this pathway are useful for various amino acids and nucleic acids. And so there's lots of things that can come from the TCA cycle that cells can find useful to do, not just catabolism, but also anabolic processes. Now, the way the TCA cycle works, though, is that there's actually an issue if you want to use the intermediates from the TCA cycle to make stuff. And so what is that issue? Well, the TCA cycle functions at a site as a cycle. And so, if we're going to take things in and out of it, that has consequences for how the cycle runs. Now there's a couple words for this that I want to just introduce to you. The first one is cataplerosis and the second one is anaplerosis. And so cataplerosis is the act of removing stuff from a metabolic cycle. So, we're going to remove citrate from the cycle to make fat. That's cataplerosis. And anaplerosis is adding stuff back to a metabolic cycle so it can continue to function. Viewing this is really evident if you think of the TCA cycle as a chicken and egg problem. So, the very first time acetyl-CoA was generated, how do you start the TCA cycle in the first place? You can't add it to the TCA cycle unless you have oxaloacetate to combine with the acetyl-CoA, which can then generate another oxaloacetate. So where does the first oxaloacetate come from? Well, we already talked about one reaction. We talked about it in the context of gluconeogenesis. That can solve this problem.

~38:47And so we have pyruvate. And so we talked earlier today how we can do oxidative decarboxylation of pyruvate to give acetyl-CoA. But we talked to in the gluconeogenesis lecture how we can add a CO2 pyruvate to generate oxaloacetate. So if I do those two reactions, now I have all the carbon I need to generate a citrate and start off the TCA cycle. Now obviously, if I do cataplerosis and I remove that citrate I made to make fat, well, now I need two pyruvate again to generate the next citrate if I'm using this pathway, because every time I bring an acetyl-CoA into the cycle, I need an oxaloacetate to combine it with. And so if I remove something, I have to add something back. And so pyruvate to oxaloacetate, the pyruvate carboxylase reaction is an example of an anaplerotic reaction. Now what this means, though, is that in order to do an anaplerosis, you have to be able to generate four-carbon oxaloacetate, or a four-carbon molecule. Now, pyruvate carboxylase, pyruvate to oxaloacetate, allows you to do that. We can take a three-carbon molecule and generate four-carbon oxaloacetate. However, if we start from a two-carbon molecule like acetate or acetyl-CoA that enters the cycle, it's actually not so simple to take that two-carbon molecule and turn it into four-carbon oxaloacetate. And in fact, humans lack any enzymes that allow them to take a two-carbon unit, to take acetate or acetyl-CoA, and turn it into anything that's longer than net-- turn it into anything that's longer than two carbons. And this has important implications for human physiology, because what it says is that we can't make glucose from anything that starts with something less than three carbons long. So if you drink alcohol and you metabolize that alcohol to acetate, it turns out you take fat and you break down fat also to acetyl-CoA, to acetate two carbons long. There is no way to turn those molecules into glucose, because you cannot generate the oxaloacetate to do the anaplerosis that's necessary to get it there. What this means is that our body can only store calories that come from two carbon units, fat or alcohol, as fat. We can never turn them back into glucose or make glycogen. And this is very relevant for those of you that go to medical school, because it's relevant to our physiology. And that is, when our bodies exhaust all of our stores of glucose, what happens? Our liver can no longer do gluconeogenesis. And so what happens? Now it has to switch over to doing something else. It has to work with two carbon units. And ultimately, this is ketone metabolism, which we'll talk about in a few lectures. It also said that the body-- that this is also the basis of a very old adage that's out there that some of you may have heard-- that you need to have some other fuel if you're going to burn fat. The basis for that is that fat is turned into two carbon units, acetyl-CoA units. And so if you're going to take those acetyl-CoA units and ultimately burn them away, turn them into CO2, you need a source of oxaloacetate, or your TCA cycle won't work. And you don't need a lot of something, but it is true. You can't start just with acetyl-CoA as a human and turn it into CO2, so you need some-- at least a little bit of oxaloacetate to get your TCA cycle started. Now, that's a problem that we as humans and other mammals face, but it turns out there's lots of microbes out there that grow just fine on acetate or on alcohol, even if it's only carbon source. And so those organisms must have some way to build stuff from two carbon units. That is a way to use two carbon units and do an anaplerosis. And it turns out the way they do this is via something called the glyoxylate cycle.

~43:06And so the glyoxylate cycle is an alternative version of the TCA cycle that effectively uses two enzymes that we lack as mammals. And so I'll quickly tell you about it here. So, this is isocitrate from the TCA cycle. And some microbes have an enzyme called isocitrate lyase. And what isocitrate lyase does is basically splits citrate in half, such that the top portion of the molecule is another TCA cycle intermediate, succinate. And the bottom portion of a molecule is this two carbon aldehyde called glyoxylate. Glyoxylate can react with acetyl-CoA by another enzyme that we lack as humans called malate synthase. And I don't have time to show the mechanism again, but malate synthase basically adds the two carbons from acetyl-CoA to the aldehyde, the carbonyl, the aldehyde carbonyl of glyoxylate in a reaction that is, for all intents and purposes, exactly what happens in citrate synthase. That will generate this molecule, which is malate, also in the TCA cycle. And so having these two extra reactions, isocitrate lyase and malate synthase, gives microbes the ability to have acetyl-CoA be anaplerotic. And so how does that work? Well, that's because if we start with one oxaloacetate, four carbons, and acetyl-CoA, two carbons, can run the TCA cycle and make citrate, six carbons. Turn that citrate into isocitrate. Use isocitrate lyase to generate glyoxylate, two carbons. Plus succinate. That succinate can run through succinate dehydrogenase to generate malate, which you can go through malate dehydrogenase to generate oxaloacetate. This glyoxylate can start with a second acetyl-CoA. Two carbons come together, generate malate. That generates a second malate molecule, which can then exit the cycle as malate or oxaloacetate or whatever you want. And so basically, it allows two acetyl-CoAs to net generate an oxaloacetate, and so net generates a way to do an anaplerosis from two carbon units by having this malate synthase reaction and this isocitrate lyase reaction and run this alternative version of the TCA cycle, called the glyoxylate cycle. And it's a nice way how life-- again, no new chemistry here, just variations on what we've already shown-- repurposed the similar chemistries that it's already using as a way to live off of carbon sources that contain only two carbons, like ethanol or acetate. So in closing today, the last thing I want to talk about, very briefly, is how the TCA cycle is regulated. And we don't need to spend a ton of time on this, because it really follows principles that make sense, particularly when we think about things that we've already talk about. And so, regulation of the TCA cycles is of course important, and it's really a critical hub, both for anabolic and catabolic pathways. So, you needed to get energy to fully oxidize carbon, but it's also a useful place to get stuff. And before I talk about regulation, I just want to point out that many of the enzymes in the TCA cycle, even though we talk about it in the mitochondria and we're about to talk about regulation in terms of catabolism-- that is oxidation, ways to release energy-- many of these enzymes are also in other locations in the cell, because there's functions for them in making stuff that is very different than what goes on in the TCA cycle. And the regulation that I'll tell you about, and that comes up on MCAT exams and stuff like that, usually talks about this pathway as a catabolic pathway, as a way to make CO2, to make ATP. However, recognize that there's also variations on this pathway that can use in anabolic things, making stuff. And that regulation is something that really is not necessarily what we're going to talk about here and is a little bit less well-understood.

~47:10But at least the regulation, in terms of catabolism, if we put it in context of glucose metabolism-- so here's glycolysis, turning glucose into pyruvate. And then that pyruvate can operate through the pyruvate dehydrogenase reaction to generate acetyl-CoA. That acetyl-CoA can combine with oxaloacetate to generate citrate. That citrate, of course, can be used to generate fat, as we've talked about. That can go to isocitrate. Isocitrate to alpha ketoglutarate. That's catalyzed by isocitrate dehydrogenase, which I will abbreviate IDH. Alpha ketoglutarate to succinate. More correctly, to succinyl-CoA by alpha ketoglutarate dehydrogenase, Alpha KGDH. And then this back to oxaloacetate. And so the main enzymes that are typically discussed as being regulated are alpha ketoglutarate dehydrogenase, isocitrate dehydrogenase, and pyruvate dehydrogenase. I also drew glycolysis. And I'll just write over here, gluconeogenesis up here, because we can now see more fully how this plays in with citrate acting as a positive regulator with gluconeogenesis and an inhibitor of glycolysis. Again, we're full in citrate, stop running glycolysis, start running gluconeogenesis. Now, the other regulation. If you have a lot of acetyl-CoA, stop making it from pyruvate. acetyl-CoA is a negative regulator of PDH. This is carbon oxidation. Releases a lot of energy, generates a lot of ATP, generates a lot of NADH. If you have lots of those things, no reason to keep sending carbon into acetyl-CoA to go into the TCA cycle. Ultimately, we have to send those electrons somewhere. So if there's nowhere to put them, low oxygen also inhibits pyruvate dehydrogenase. And of course, if you need more energy, if ADP is high, that activates pyruvate dehydrogenase. So at least in terms of glucose, complete glucose oxidation, a lot of regulation happens at pyruvate dehydrogenase. And a lot of it really makes sense. High levels of ATP, high levels of NADH, also inhibit isocitrate dehydrogenase and alpha ketoglutarate dehydrogenase. Makes sense. Succinol-CoA high, inhibit alpha ketoglutarate dehydrogenase. High levels of ADP, need to release more energy, activate alpha ketoglutarate dehydrogenase. Again, these are the feedbacks that people talk about on board exams. Good to know, but you can almost guess what they would be from first principles. Because remember, this is a pathway that releases a lot of energy. High energy high ATP, high NADH-- don't run the cycle, don't enter carbon in the cycle. Low energy, high ADP, put carbon in the cycle, run the cycle faster. Makes sense. Also makes sense of the reciprocal regulation of glycolysis and gluconeogenesis. Great. Next time, we will talk more about how we can oxidize fatty acids, fat, by accessing acetyl-CoA and entering it into the cycle. Thanks.

★本章小结：核心代谢逻辑链

1. 糖异生的热力学绕道与跨膜协同逻辑：糖酵解的 3 步大放能不可逆反应（HK、PFK-1、PK）决定了逆向合成葡萄糖必须另辟蹊径。细胞在肝脏（90%）和肾皮质（10%）中跨线粒体、细胞质和内质网三个区室，动用 4 种特有关键酶——① 丙酮酸羧化酶（PC，线粒体，辅基生物素，耗 ATP，必需乙酰-CoA 别构激活）将丙酮酸羧化为草酰乙酸（经苹果酸穿梭出线粒体并顺带把 NADH 搬运到胞质），再由 ② PEPCK（耗 GTP）利用 β-酮酸自发脱羧释放的自由能驱动合成高能 PEP（脱去的正是刚加上的那个 CO₂）；③ FBPase-1 与 ④ G-6-Pase（仅肝、肾、肠内质网有，肌肉缺乏）分别通过不可逆水解绕过 PFK-1 和 HK。从 2 丙酮酸合成 1 葡萄糖共耗 4 ATP + 2 GTP（6 个高能键）+ 2 NADH，与肌肉酵解配合构成 Cori 循环（每次净耗 4 个高能键）及葡萄糖-丙氨酸循环（靠 ALT 既运碳又运氨）。
2. 糖原高效存取与双刃剑激素调控逻辑：糖原分解采用糖原磷酸化酶（GP，辅基 PLP）催化非还原端 α-1,4 键的磷酸解（生成 90% G-1-P，省下 1 ATP 且带电锁在胞内），配合双功能脱支酶（先转移 3 个残基、再水解 α-1,6 键释放 10% 游离葡萄糖）和磷酸葡糖变位酶（Ser-P）；糖原合成则通过 UDPG 焦磷酸化酶消耗 UTP 生成活性供体 UDPG（靠焦磷酸水解拉动），在生糖原蛋白（Glycogenin Tyr194 自糖基化）引物基础上，由糖原合酶（GS）延伸 α-1,4 主链、分支酶建立 α-1,6 分支。肾上腺素/胰高血糖素经 cAMP→PKA 触发磷酸化，同时激活 GP（b→ a）并灭活 GS（a→ b）；胰岛素则通过激活 PP1 和抑制 GSK3 实现反向去磷酸化调控。
3. PDH 复合体与 TCA 循环的氧化能量中枢逻辑：丙酮酸经内膜载体进入线粒体基质，由 PDH 复合体（E1、E2、E3 三酶与 TPP、硫辛酸、CoA、FAD、NAD⁺ 五辅酶）通过 E2 硫辛酰胺摆臂底物通道催化不可逆氧化脱羧生成乙酰-CoA 和 NADH（亚砷酸盐螯合二氢硫辛酰胺双巯基而致死）。随后乙酰-CoA 进入 TCA 循环经 8 步反应（柠檬酸合酶诱导契合防乙酰-CoA 水解 → 顺乌头酸酶前手性专一识别草酰乙酸支臂并受氟柠檬酸“致死合成”抑制 → IDH 与 α-KGDH 两步氧化脱羧释放完全来自草酰乙酸的 2 CO₂ → 琥珀酰-CoA 合成酶唯一底物水平磷酸化产 1 GTP → 唯一内膜酶琥珀酸脱氢酶产 1 FADH₂ 并受丙二酸竞争抑制 → 延胡索酸酶反式水合 → 苹果酸脱氢酶克服 +29.7 kJ/mol 能障再生 OAA），每轮产 3 NADH + 1 FADH₂ + 1 GTP = 10 ATP，使 1 分子葡萄糖彻底有氧氧化净产 30 或 32 ATP（旧口径 36/38 ATP）。
4. 碳骨架收支平衡与化脂为糖逻辑：TCA 循环兼具分解与合成两用性；当草酰乙酸等中间物被抽走用于合成时，动物主要靠丙酮酸羧化酶进行回补反应。由于 PDH 不可逆且 TCA 每进 2C 必丢 2 CO₂，动物绝不能将乙酰-CoA（偶数碳脂肪酸）净转化为糖；唯有植物和部分微生物凭借乙醛酸循环体中的异柠檬酸裂解酶和苹果酸合酶绕过两次脱羧，将 2 分子乙酰-CoA 净合成 1 分子四碳二羧酸进入糖异生，实现油料种子萌发时的“化脂为糖”。

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★名词解释速背卡（中英对照高频名解）

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★易混概念与新旧教材口径辨析

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★华农历年真题章末综合自测