1. Citric Acid Cycle

2. Figure 17-2 Citric Acid Cycle

3. Summary of Citric Acid Cycle Acetyl-CoA + 3 NAD + + FAD + GDP + P i 2 CO 2 + 3 NADH + 3H + + FADH 2 + GTP + CoA-SH

4. Reactions of the Citric Acid Cycle

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

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

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

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

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

10. Aconitase Stereospecific Addition ∆G o ’ = ~0

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

12. Page 325 Stereospecificity of Aconitase Reaction Prochiral SubstrateChiral Product

13. Figure 11-2 Stereospecificity in Substrate Binding

14. NAD + –Dependent Isocitrate Dehydrogenase Oxidative Decarboxylation NOTE: CO 2 from oxaloacetate ∆G o ’ = -20.9 kJ/mol

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

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

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

18. Regulation of Isocitrate 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 CO 2 Isocitrate dehydrogenase CO 2 Isocitrate dehydrogenase

20.  -Ketoglutarate Dehydrogenase Oxidative Decarboxylation Mechanism similar to PDH CO 2 from oxaloacetate High energy thioester ∆G o ’ = -33.5 kJ/mol

21.  -Ketoglutarate Dehydrogenase (Multienzyme Complex) E 1 :  -Ketoglutarate Dehydrogenase or  -Ketoglutarate Decarboxylase E 2 : Dihydrolipoyl Transsuccinylase E 3 : Dihydrolipoyl Dehydrogenase (same as E 3 in PDH)

22. Regulation of  -Ketoglutarate Dehydrogenase Inhibitors –NADH –Succinyl-SCoA Activator: Ca 2+

23. Origin of C-atoms in CO 2 Both CO 2 carbon atoms derived from oxaloacetate

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

25. Thermodynamics (Succinyl-SCoA Synthetase)

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

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

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

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

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

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

32. Figure 17-13 Covalent Attachment of FAD

33. FAD used for Alkane  Alkene Reduction Potential –Affinity for electrons; Higher E, Higher Affinity –Electrons transferred from lower to higher E  E h o’ =  G o’ /nF = -(RT/nF)ln (K eq ) FAD/FADH 2 Succinate/Fumarate NAD+/NADH Isocitrate/α-Ketoglutarate Reduction Potential

34. Fumarase ∆G o ’ = ~0

35. Page 583 Mechanism of Fumarase

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

37. Thermodynamics

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

39. Page 584 ATP Production from Products of the Central metabolic Pathway = 32 ATP NADH  2.5 ATP FADH 2  1.5 ATP

40. Amphibolic Nature of Citric Acid Cycle

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

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

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 CO 2 Each turn after will lose ½ remaining radioactivity

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

46. You are following the metabolism of pyruvate in which the methyl-carbon is radioactive: *CH 3 COCOOH. -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 (CO 2 Carrier)

51. Reaction Mechanism I (Dehydration/Activation of HCO 3 – )

52. Reaction Mechanism II (Transfer of CO 2 to Pyruvate)

53. Fates of Oxaloacetate Regulation!

54. Regulation of Pyruvate Carboxylase Allosteric Activator Acetyl-SCoA

55. Glyoxylate Cycle Glyoxysome Plants and Some Microorganisms

56. Citrate Synthase (citrate condensing enzyme)

57. Aconitase

58. Glyoxylate Cycle Enzymes Plants and Some Microorganisms

59. Malate Dehydrogenase

60. Net Reaction of Glyoxylate Cycle Net increase of one 4-carbon unit! 2 Acetyl-CoA  1 Oxaloacetate

61. Figure 17-18 Glyoxylate Cycle and the Glyoxysome

62. Regulation of the Citric Acid Cycle

63. Regulatory Mechanisms Availability of substrates –Acetyl-CoA –Oxaloacetate –Oxygen (O 2 ) Need for citric acid cycle intermediates as biosynthetic precursors Demand for ATP

64. Table 17-2 Free Energy Changes of Citric Acid Cycle Enzymes

65. Regulation of Pyruvate Dehydrogenase Product Inhibition (competitive) –NADH –Acetyl-SCoA Phosphorylation/Dephosphorylation –PDH Kinase: inactivation –PDH Phosphatase: reactivation

66. Figure 17-15 Covalent Modification and Regulation of PDH

67. Regulation of PDH Kinase (Inactivation) Activators –NADH –Acetyl-SCoA Inhibitors –Pyruvate –ADP –Ca 2+ (high Mg 2+ ) –K +

68. Regulation of PDH Phosphatase (Reactivation) Activators –Mg 2+ –Ca 2+

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

70. Regulation of Isocitrate Dehydrogenase Pulls aconitase reaction Regulation (allosteric enzyme) –Positive Effector: ADP (energy charge) –Negative Effector: ATP (energy charge) Accumulation of Citrate: inhibits Phosphofructokinase

71. Regulation of  -Ketoglutarate Dehydrogenase Inhibitors –NADH –Succinyl-SCoA Activator: Ca 2+

72. Figure 17-16 Regulation of the Citric Acid Cycle