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Citric Acid Cycle

Citric Acid Cycle. Citric Acid Cycle. Figure 17-2. Summary of Citric Acid Cycle. Acetyl-CoA + 3 NAD + + FAD + GDP + P i. 2 CO 2 + 3 NADH + 3H + + FADH 2 + GTP + CoA-SH. Reactions of the Citric Acid Cycle. Citrate Synthase (citrate condensing enzyme). ∆G o ’ = –31.5 kJ/mol.

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Citric Acid Cycle

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  1. Citric Acid Cycle

  2. Citric Acid Cycle Figure 17-2

  3. Summary of Citric Acid Cycle Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi 2 CO2 + 3 NADH + 3H+ + FADH2 + GTP + CoA-SH

  4. Reactions of the Citric Acid Cycle

  5. Citrate Synthase(citrate condensing enzyme) ∆Go’ = –31.5 kJ/mol

  6. Mechanism of Citrate Synthase(Formation of Acetyl-SCoA Enolate) Figure 17-10 part 1

  7. Mechanism of Citrate Synthase(Acetyl-CoA Attack on Oxaloacetate) Figure 17-10 part 2

  8. Mechanism of Citrate Synthase(Hydrolysis of Citryl-SCoA) Figure 17-10 part 2

  9. Regulation of Citrate Synthase • Pacemaker Enzyme (rate-limiting step) • Rate depends on availability of substrates • Acetyl-SCoA • Oxaloacetate

  10. Aconitase ∆Go’ = ~0 Stereospecific Addition

  11. Iron-Sulfur Complex(4Fe-4S] Thought to coordinate citrate –OH to facilitate elimination

  12. Stereospecificity of Aconitase Reaction Prochiral Substrate Chiral Product Page 325

  13. Stereospecificity in Substrate Binding Figure 11-2

  14. NAD+–DependentIsocitrate Dehydrogenase ∆Go’ = -20.9 kJ/mol Oxidative Decarboxylation NOTE: CO2 from oxaloacetate

  15. Mechanism of Isocitrate Dehydrogenase(Oxidation of Isocitrate) Figure 17-11 part 1

  16. Mechanism ofIsocitrate Dehydrogenase(Decarboxylation of Oxalosuccinate) Mn2+ polarizes C=O Figure 17-11 part 2

  17. Mechanism ofIsocitrate Dehydrogenase(Formation of -Ketoglutarate) Figure 17-11 part 2

  18. Regulation ofIsocitrate Dehydrogenase • Pulls aconitase reaction • Regulation (allosteric enzyme) • Positive Effector: ADP (energy charge) • Negative Effector: ATP (energy charge) • Accumulation of Citrate: inhibits Phosphofructokinase

  19. Accumulation of Citrate CO2 CO2 Isocitrate dehydrogenase Isocitrate dehydrogenase

  20. -Ketoglutarate Dehydrogenase ∆Go’ = -33.5 kJ/mol Oxidative Decarboxylation Mechanism similar to PDH CO2 from oxaloacetate High energy thioester

  21. a-Ketoglutarate Dehydrogenase(Multienzyme Complex) • E1: -Ketoglutarate Dehydrogenase or -Ketoglutarate Decarboxylase • E2: Dihydrolipoyl Transsuccinylase • E3: Dihydrolipoyl Dehydrogenase (same as E3 in PDH)

  22. Regulation of-Ketoglutarate Dehydrogenase • Inhibitors • NADH • Succinyl-SCoA • Activator: Ca2+

  23. Origin of C-atoms in CO2 Both CO2 carbon atoms derived from oxaloacetate

  24. Succinyl-CoA Synthetase(Succinyl Thiokinase) ∆Go’ = ~0 High Energy Thioester —> Phosphoanhydride Bond Plants and Bacteria: ADP + Pi —> ATP Randomizationn of labeled C atoms

  25. Thermodynamics(Succinyl-SCoA Synthetase)

  26. Evidence for Phosphoryl-enzyme Intermediate(Isotope Exchange) Absence of Succinyl-SCoA Page 581

  27. Mechanism ofSuccinyl-CoA Synthetase(Formation of High Energy Succinyl-P) Figure 17-12 part 1

  28. Mechanism ofSuccinyl-CoA Synthetase(Formation of Phosphoryl-Histidine) Figure 17-12 part 2

  29. Mechanism ofSuccinyl-CoA Synthetase(Phosphoryl Group Transfer) Substrate-level phosphorylation Figure 17-12 part 3

  30. Nucleoside Diphosphate Kinase(Phosphoryl Group Transfer) GTP + ADP ——> GDP + ATP ∆Go’ = ~0

  31. Succinate Dehydrogenase ∆Go’ = ~0 Randomization of C-atom Labeling Membrane-Bound Enzyme

  32. Covalent Attachment of FAD Figure 17-13

  33. FAD used for Alkane Alkene • Reduction Potential • Affinity for electrons; Higher E, Higher Affinity • Electrons transferred from lower to higher E Eho’ = Go’/nF = -(RT/nF)ln (Keq) Reduction Potential

  34. Fumarase ∆Go’ = ~0

  35. Mechanism of Fumarase Page 583

  36. Malate Dehydrogenase ∆Go’ = +29.7 kJ/mol Low [Oxaloacetate]

  37. Thermodynamics

  38. Products of the Citric Acid Cycle Figure 17-14

  39. ATP Production from Products of the Central metabolic Pathway = 32 ATP NADH  2.5 ATP FADH2 1.5 ATP Page 584

  40. Amphibolic Nature of Citric Acid Cycle

  41. 1 2 3 6 5 4 Carbons of Glucose:1st cycle 3, 4 2,5 1,6 2,5 1,6 1,6 2,5 2,5 1,6 1,6 2,5

  42. Carbons of Glucose:2nd cycle:Carbons 2,5:After 1½ turns:all radioactivity is CO2

  43. Carbons of Glucose:2nd cycle:Carbons 1,6:After 2 turns:¼ radioactivity in each carbon of OAA

  44. Carbons of Glucose:3rd cycle:Carbons 1,6:After 3 turns:½ radioactivity is CO2Each turn after willlose ½ remainingradioactivity

  45. Carbon Tracing from Glucose • Glucose radiolabeled at specific Carbons • Can determine fate of individual carbons • Carbons 1,6 • 1st cycle: 1, 4 of oxaloacetate • Starting at 3rd cycle ½ radioactivity  CO2/cycle • Carbons 2,5 • 1st cycle: 2, 3 of oxaloacetate • 2nd cycle: all eliminated as CO2 • Carbons 3,4 • All eliminated at CO2 during Pyruvate  Acetyl-CoA

  46. You are following the metabolism of pyruvate in which the methyl-carbon is radioactive: *CH3COCOOH. -assuming all the pyruvate enters the TCA cycle as Acetyl-CoA, indicate the labeling pattern and its distribution in oxaloacetate first formed by operation of the TCA cycle.

  47. Generation of Citric Acid Cycle Intermediates

  48. Pyruvate Carboxylase Mitochondrial Matrix

  49. Pyruvate Carboxylase Animals and Some Bacteria

  50. Biotin Cofactor(CO2 Carrier)

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