第20章 柠檬酸循环.ppt

第20章 柠檬酸循环,主要内容,TCA准备阶段TCA循环阶段TCA产能计算TCA的调节TCA双重作用,TCA背景知识,1、为什么称为柠檬酸循环、三羧酸循环（Tricarboxylic acid cycle, TCA） 、Krebs循环？ 在有氧的情况下，葡萄糖酵解产生的丙酮酸氧化脱羧形成乙酰CoA乙酰CoA经一系列氧化、脱羧，最终生成C2O和H2O并产生能量的过程，称为柠檬酸循环，亦称为三羧酸循环(tricarboxylic acid cycle), 简称TCA循环由于它是由H.A.Krebs（德国）正式提出的，所以又称Krebs循环It is convenient to use a brief term for the kind of scheme. Its essential feature is the periodic formation of a number of di- and tricarboxylic acids. As there is no term which would serve as a common denominator for all the various acids, it seemed reasonable to name the cycle,after one, or some, of its characteristic and specific acids. It was from such considerations that the term "citric acid cycle" was proposed in 1937.” （Hans A. Krebs, The citric acid cycle, Nobel Lecture, December 11, 1953）,Krebs, 1901-1981,Brief history of TCA,The first major investigation into the intermediary metabolism of oxidation was that of Thunberg, who examined systematically the oxidizability of organic substances in isolated animal tissues. Between 1906 and 1920 he tested the oxidation of over 60 organic substances, chiefly in muscle tissue. He discovered the rapid oxidation of the salts of a number of acids, such as lactate(乳酸盐), succinate(琥珀酸盐), fumarate(延胡索酸盐), malate(苹果酸盐), citrate(柠檬酸盐), and glutamate(谷氨酸盐).,In 1932, Krebs was studying the rates of oxidation of small organic acids by kidney and liver tissue. Only a few of substances were active in these experiments---notably succinate, fumarate, acetate, malate, and citrate.,An important development came from the laboratory of Szent-Gyorgyi of Szegedin 1935, who confirmed on pigeon breast muscle the rapid oxidation of the C4-dicarboxylic acids - succinic, fumaric, malic, and oxaloacetic acids - and arrived at the new conclusion that those dicarboxylic acids were linked by an enzymatic pathway that was important for aerobic metabolism.,A decisive contribution to the field was made in March 1937 by Martius and Knoop, who discovered α-ketoglutarate( α- 酮戊二酸) as a product of citrate oxidation. Because it was already known that α- ketoglutarate could be enzymatically oxidized to succinate, the pathway from citrate to oxaloacetate was seemed as:,In 1937, Krebs found that citrate could be formed in muscle suspensions if oxaloacetate and either pyruvate or acetate were added. Now, he get a cycle:,TCA背景知识,2、细胞呼吸（cell respiration）要经历三个阶段：糖酵解阶段、柠檬酸循环阶段、氧化磷酸化阶段。

3、糖酵解的产物丙酮酸进入TCA之前有一准备过程，即形成乙酰CoATCA准备阶段,丙酮酸在丙酮酸脱氢酶系催化下氧化脱羧形成乙酰辅酶A丙酮酸脱氢酶系复合物含三种酶和五个辅助因子,E1: pyruvate dehydrogenase, thiamine pyrophosphate (TPP)E2: dihydrolipoyl transacetylase, lipoic acid, coenzyme A-SH E3: dihydrolipoyl dehydrogenase, NAD+, FAD,IRREVERSIBLE,TCA准备阶段,,,H+,,,丙酮酸脱氢酶系作用机理（1）,1. 丙酮酸与TPP结合并 脱羧形成羟乙基TPP2. 羟乙基TPP氧化转变成 乙酰基同时转移到E2的 辅基硫辛酰胺上3. 在E2上的乙酰基在E2 催化下转移到CoASH 上形成游离的乙酰CoA. 从而形成了一个高能硫 酯键The mechanistic details of the first three steps of the pyruvate dehydrogenase complex reaction,TCA准备阶段,,TCA准备阶段,,,丙酮酸脱氢酶系作用机理（2）,4. 还原型的E2将二个SH基H 转移到E3的辅酶FAD上形 成还原型FADH,5. E3上的还原型的FADH将 H交给NAD+形成NADH， E3辅基又形成氧化型的 FAD,,,TCA准备阶段,,,丙酮酸脱氢酶系反应图解,E1,E3,TCA准备阶段,砷化物对硫辛酰胺的毒害作用 有机砷化物和亚砷酸能与丙酮酸脱氢酶系中的E2辅基硫辛酰胺共价结合，使还原型的硫辛酰胺形成失去催化能力的砷化物。

这类砷化物同样表现在对酮戌二酸脱氢酶系的抑制上TCA概貌,,,2C,6C柠檬酸,6C异柠檬酸,5C 酮戌二酸,4C琥珀酰CoA,4C琥珀酸,4C延胡索酸,4C苹果酸,4C草酰乙酸,1、草酰乙酸与乙酰辅酶A形成柠檬酸—催化此反应的酶为柠檬酸合酶；—反应的中间产物为柠檬酰辅酶A；—柠檬酸合酶属于调控酶，其活性受ATP、NADH、琥珀酰CoA、酯酰CoA等的抑制；另一种抑制剂是丙酮酰CoA—它是TCA循环的限速酶，由氟乙酸形成的氟乙酰CoA可被该酶催化形成氟柠檬酸，从而抑制下一步的顺乌头酸酶催化的反应此称为致死性合成反应TCA循环阶段,,S-CoAintermediate,哺乳动物体内柠檬酸合成酶以二聚体形式存在与草酰乙酸的结合使酶发生有利于和乙酰CoA结合的形变TCA循环阶段,,柠檬酸合成酶的单聚体形式，绿色原子为柠檬酸，粉色原子为CoA,TCA循环阶段,,草酰乙酸与乙酰辅酶A形成柠檬酸，反应的中间产物为柠檬酰辅酶A２、柠檬酸异构形成异柠檬酸—催化此反应的酶为乌头酸酶；—反应的中间产物为顺乌头酸；—反应为先脱水后水化；—由于反应生成的异柠檬酸在下一步反应中迅速被氧化而使反应向生成异柠檬酸的方向进行。

TCA循环阶段,,顺乌头酸酶催化柠檬酸异构化为柠檬酸，反应分两步进行，经历一个顺乌头酸中间体反应具有严格的空间特异性TCA循环阶段,,,顺乌头酸酶活性位点的铁硫聚簇TCA循环阶段,,氟乙酸到氟柠檬酸的转化,TCA循环阶段,,３、异柠檬酸氧化生成－酮戌二酸—催化此反应的酶为异柠檬酸脱氢酶；—反应为TCA二次氧化脱羧中的第一个反应；—反应中间产物为不稳定的草酰琥珀酸；—既有以NAD+为辅酶的异柠檬酸脱氢酶，也有以NADP+为辅酶的异柠檬酸脱氢酶—异柠檬酸脱氢酶是变构调节酶，其活性受ADP和NAD+的变构激活，受ATP和NADH的变构抑制TCA循环阶段,,-脱羧反应,TCA循环阶段,,4、－酮戌二酸氧化脱羧生成琥珀酰CoA—催化此反应的酶为－酮戌二酸脱氢酶复合体，该酶由－酮戌二酸脱氢酶E1、二氢硫辛酰转琥珀酰酶E2和二氢硫辛酰脱氢酶E3及六种辅助因子TPP、硫辛酸、CoA、NAD+、FAD、Mg2+组成；—反应为TCA二次氧化脱羧中的第二个反应；—反应释放的能量主要存于琥珀酰CoA的高能硫酯键中；,TCA循环阶段,,4、－酮戌二酸氧化脱羧生成琥珀酰CoA —－酮戌二酸脱氢酶是变构调节酶，其活性受产物琥珀酰CoA、NADH和高能ATP的变构抑制。

—与丙酮酸脱氢酶复合体中E1不同的是该酶不受磷酸化与去磷酸化的共价修饰调节作用；,TCA循环阶段,,5、琥珀酰CoA转化为琥珀酸并释放高能磷酸键—催化此反应的酶为琥珀酰CoA合成酶或称琥珀酰硫激酶；—该反应为TCA是唯一直接产生高能磷酸键的步骤，也是一步底物水平磷酸化产生能量的步骤；—反应生产的GTP在蛋白质的生物合成中起磷酰基供体及激活信号蛋白的作用，也可以与ADP磷酸化生成ATP相偶联产生能量TCA循环阶段,,琥珀酰CoA合成酶反应机制,TCA循环阶段,,6、琥珀酸脱氢生成延胡索酸—催化此反应的酶为琥珀脱氢酶；它以FAD为辅基；—该酶具有严格的立体专一性，即只生成反式延胡索酸；—与琥珀酸结构类似的化合物如丙二酸、戌二酸等是该酶的竞争性抑制剂TCA循环阶段,,TCA循环阶段,,琥珀酸脱氢酶的铁硫聚簇,FAD和琥珀酸脱氢酶的共价结合,7、延胡索酸水合成L-苹果酸—催化此反应的酶为延胡索酸酶；—该酶具有严格的立体专一性，即只生成L-苹果酸；,TCA循环阶段,,TCA循环阶段,,延胡羧酸酶的两种可能的反应机制,8、L-苹果酸脱氢生成草酰乙酸—催化此反应的酶为苹果酸脱氢酶；—该酶的辅基为NAD+；—由于草酰乙酸与乙酰CoA合成柠檬酸的反应是高度放能反应，因此通过草酰乙酸的不断消耗来驱使该反应不断向生成草酰乙酸方向进行。

TCA循环阶段,,TCA循环阶段,,以NAD+作为辅酶的脱氢酶的空间特异性比较,TCA循环阶段,,苹果酸脱氢酶的结构,TCA循环阶段,,TCA Cycle,乙酰草酰成柠檬，柠檬易成α-酮琥酰琥酸延胡索，苹果落在草丛中。