生物化學教學課件：lecture3Citric Acid Cycle（TCA)

1、Lecture3 Citric Acid Cycle Tricarboxylic acid cycle (TCA cycle or Krebs cycle) 生物化學教程 p332-347HANS ADOLF KREBS 1953 Nobel Laureate in Medicine for his discovery of the citric acid cycle Background 1900-1981Place of Birth: Hildesheim, GermanyResidence: Great BritainAffiliation: Sheffield University H

2、ans Krebs, 190019811 Production of Acetyl-CoA2 Reactions of the Citric Acid Cycle3 Regulation of the Citric Acid Cycle 4 The Glyoxylate Cycle(乙醛酸循環(huán)）乙醛酸循環(huán)）outlineTCA Take place Eukaryotes: mitochondria Prokaryotes: cytosolcellular respiration -Rather than being reduced to lactate, ethanol, or some ot

3、her fermentation product, the pyruvate produced by glycolysis is further oxidized to H2O and CO2. 重點重點1. Production of Acetyl-CoA -Pyruvate Oxidation A Major Entry Route for Carbon into the Citric Acid Cycle Pyruvate Dehydrogenase Complex 丙酮酸脫氫酶系丙酮酸脫氫酶系重點重點Pyruvate Dehydrogenase Complex enzymes(E1,E

4、2 and E3):1. Pyruvate Dehydrogenase (E1)2. Dihydrolipoyl transacetylase (E2)二氫硫辛酰轉(zhuǎn)乙?；?. Dihydrolipoyl dehydrogenase (E3)二氫硫辛酰脫氫酶five coenzymes:1. Thiamine Pyrophosphate 焦磷酸硫胺素, 輔羧酶(TPP)2. Lipoic Acid硫辛酸- lipoamide硫辛酰胺3. FAD4. NAD+5. CoASH重點重點TPP is the coenzyme form of vitamin B1 (thiamine)catalyze

5、d by Pyruvate Dehydrogenase , two of the three carbons of pyruvate are carried transiently on TPP in the form of a hydroxyethyl 羥乙基羥乙基or “active acetaldehyde乙醛乙醛” group which is subsequently released as acetaldehydeThe reactive carbon atom羥乙基羥乙基TPP（HETPP)焦磷酸硫胺素The TPP carbanion負碳離子acts as a nucleoph

6、ile親核, attacking the carbonyl group of pyruvate.Decarboxylation produces a carbanion負碳離子 that is stabilized by the thiazolium噻唑 ring. Protonation to form hydroxyethyl TPP release of acetaldehyde. A proton dissociates to regenerate the carbanion.strong tendency to form a new bondis generally unstable

7、羥乙基羥乙基TPPCryoelectron冷凍電子顯微鏡術 micrograph of PDH complexes isolated from bovine kidney.Three dimensional image of PDH complexThe core (green) consists of 24 molecules of E2, arranged in 8 trimers The lipoyl 硫辛?；?domain of E2 (blue) reaches outward to touch the active sites of E1 molecules (yellow) A

8、number of E3 subunits (red) are also bound to the core, where the swinging arm on E2 can reach their active sites. 二氫硫辛酰轉(zhuǎn)乙?；付淞蛐刘Ｃ摎涿?. E1 accepts a two carbon aldehyde（乙醛） from the decarboxylation of pyruvate.2. The aldehyde group is transferred to the first lipoamide（硫辛酰胺） arm of E2.3. The acetyl

9、group is linked to CoASH, forming acetyl-CoA.4. E3 oxidizes the reduced lipoamide arm by transferring two hydrogens to FAD, forming FADH25. FADH2 is oxidized by NAD+, forming FAD and NADH + H+.central metabolic pathway中心代謝途徑中心代謝途徑1. generates NADH and FADH2 2. produces GTP via substrate-level phosph

10、orylation3. Many metabolic processes use intermediates of the TCA in their pathways. 2. Reactions of TCA重點重點cyclic pathway begin with addition of acetyl-CoA to oxaloacetate to form citrateThe TCA Has Eight StepsSteps1,3,and 4 are essentially irreversible in the cellStep5 may be either ATP or GTPFour

11、 steps in this process are oxidations, energy of oxidation - NADH and FADH2.重點重點Phase 1: Introduction and Loss of Two Carbon AtomsStep 1: Introduction of Two Carbon Atoms as Acetyl-CoA Step 2: Isomerization（異構(gòu)化） of Citrate（檸檬酸） Step 3: Generation of CO2 by an NAD+ Linked Dehydrogenase Step 4: Genera

12、tion of a Second CO2 by a Multienzyme ComplexThe Citric Acid Cycle Has Eight StepsPhase 2: Regeneration of Oxaloacetate（草酰乙酸）（草酰乙酸）Step 5: A Substrate-Level Phosphorylation Step 6: A Flavin-Dependent Dehydrogenase Step 7: Hydration 水合作用of a Carbon-Carbon Double Bond Step 8: A Dehydrogenation that Re

13、generates OxaloacetateStep 1: Introduction of Two Carbon Atoms as Acetyl-CoA檸檬酸合酶citrate synthase TCA 限速酶限速酶The large, negative standard free-energy change of the citrate synthase reaction is essential to the operation of the cycle the concentration of oxaloacetate草酰乙酸 is normally very low (10-6). 重

14、點重點Structure of citrate synthase(a) open form of the enzyme alone (b) closed form with bound oxaloacetate and a stable analog of acetyl-CoA .The flexible domain of each subunit undergoes a large conformational change on binding oxaloacetate creating a binding site for acetyl-CoA.oxaloacetateacetyl-C

15、oACitrateCitric acid is produced industrially by growing the fungus Aspergillus曲霉菌曲霉菌 niger in the presence of an inexpensive sugar source, usually beet molasses(甜菜甜菜糖蜜糖蜜). Culture conditions are designed to inhibit the reactions of the citric acid cycle such that citrate accumulates.Step 2: Isomeri

16、zation of Citrate順烏頭酸叔醇化合物仲醇化合物（可氧化）Aconitase烏頭酸酶aconitate hydratase烏頭酸水合酶烏頭酸水合酶重點重點檸檬酸檸檬酸 異檸檬酸異檸檬酸The iron-sulfur center acts in both substrate binding and catalysis.Three Cys residues of the enzyme bind three iron atoms the fourth iron is bound to one of the carboxyl groups of citrate and also int

17、eracts noncovalently with a hydroxyl group of citrate. A basic residue (:B) on the enzyme helps to position the citrate in the active site. Aconitase烏頭酸梅烏頭酸梅Step 3: Generation of CO2 by an NAD+ Linked DehydrogenaseIsocitrate dehydrogenase 異檸檬酸脫氫酶異檸檬酸脫氫酶酮酸的形成可促進臨近C-C鍵的斷裂，有利于脫羧作用的進行ADP酮酸草酰琥珀酸Coenzyme

18、NAD+ Mit coenzyme NADP+ Mit and cpATP，NADH inhibit the activity of isocitrate dehydrogenase重點重點異檸檬酸異檸檬酸 -酮戊二酸酮戊二酸Step 4 Generation of a Second CO2 by a Multienzyme Complex -KG 脫氫酶系脫氫酶系1. - - KD dehydrogenase (E1)2. Dihydrolipoamide transacetylase (E2)二氫硫辛酰二氫硫辛酰轉(zhuǎn)琥珀酰轉(zhuǎn)琥珀酰酶酶3. Dihydrolipoamide dehydroge

19、nase (E3)二氫硫辛酰脫氫酶二氫硫辛酰脫氫酶five coenzymes1. Thiamine Pyrophosphate 焦磷酸硫胺素焦磷酸硫胺素, 輔羧酶輔羧酶(TPP)2. Lipoic Acid- lipoamide硫辛酰胺硫辛酰胺3. FAD4. NAD+5. CoASH重點重點-酮戊二酸酮戊二酸 琥珀酰琥珀酰CoA Step 5: Conversion of Succinyl-CoA to SuccinatePhase 2: Regeneration of Oxaloacetate（草酰乙酸）（草酰乙酸）琥珀酰琥珀酰CoA合成酶合成酶底物水平磷酸化- Only one in

20、TCAIn animal: GTPIn plant and microorganism: ATP重點重點Different from synthase 琥珀酰琥珀酰CoA 琥珀酸琥珀酸 a phosphoryl group replaces the CoA of succinyl-CoA bound to the enzyme, forming a high-energy acyl phosphate. ?；姿狨；姿?the succinyl phosphate donates its phosphoryl group to a His residue on the enzyme, for

21、ming a high-energy phosphohistidyl enzyme the phosphoryl group is transferred from the His residue to the terminal phosphate of GDP (or ADP), forming GTP (or ATP).Step 6: Oxidation of Succinate to Fumarate琥珀酸脫氫酶succinate dehydrogenase-A Flavin-Dependent Dehydrogenase 重點重點琥珀酸脫氫酶琥珀酸脫氫酶H+H+ 琥珀酸琥珀酸 延胡索酸

22、延胡索酸 lMalonate(丙二酸丙二酸), analog of succinate, is a strong competitive inhibitor.lOnly enzyme of TCA is membrane-boundStep 7: Hydration of a Carbon-Carbon Double Bond 延胡索酸酶Step 7: Hydration of Fumarate to MalatefumaraseFumarase- highly stereospecific重點重點延胡索酸酶延胡索酸酶延胡索酸延胡索酸 L-蘋果酸蘋果酸 Step 8: Oxidation of

23、 Malate to OxaloacetateA Dehydrogenation That Regenerates Oxaloacetate 蘋果酸脫氫酶蘋果酸脫氫酶Malate dehydrogenase重點重點L-蘋果酸蘋果酸 草酰乙酸草酰乙酸 For each acetyl-CoA oxidized by the citric acid cycle, 3 NADH 1 FADH2 1 ATP or GTPThe Energy of Oxidations in the Cycle重點重點Note:A two-carbon acetyl group entered the cycle by

24、combining with oxaloacetate. Two carbon atoms emerged from the cycle as CO2 from the oxidation of isocitrate and -ketoglutarate.the two carbon atoms appearing as CO2 are not the same two carbons that entered in the form of the acetyl group重點重點Energetics of the Citric Acid Cycle10 ATP3032ATP重點重點Besid

25、es acetyl-CoAany compound that gives rise to a four- or five-carbon intermediate of the citric acid cyclecan be oxidized by the cycleThe TCA is amphibolic (雙重代謝的雙重代謝的)serving in both catabolism and anabolism-cycle intermediates can be drawn off and used as the starting material for a variety of bios

26、ynthetic products.重點重點3. Regulation of Pyruvate Dehydrogenase and the Citric Acid Cycleregulated in two primary ways controlling the entry of fuel into the cycle controlling key reactions within the cycle重點重點Pyruvate dehydrogenase - major regulatory point for entry of materials into TCAThe enzyme is

27、 regulated allosterically and by covalent modification.Allosteric RegulationE2 - inhibited by acetyl-CoA, activated by CoA-SHE3 - inhibited by NADH, activated by NAD+.ATP: allosteric inhibitor of the complexAMP: activator3 .1 Controlling the entry of fuel into the cycle重點重點Covalent Regulation (E1)重點重點3 .2 Key reactions within the cycle: allosteric regulation isocitrate dehydrogenase Activated by ADP, inhibited by NADH Phosphorylation of one serine residue in the enzyme prevents binding of isocitratel ketoglutarate dehydrogenaseInhibited by succinyl-CoA and NADH重點重點Plants some