1. CITRIC ACID CYCLE Student Edition 11/8/13 version
Dr. Brad Chazotte
213 Maddox Hall
Web Site:
Original material only © B. Chazotte
Pharm. 304 Biochemistry
Fall 2014

2. Goals
Learn the Citric Acid Cycle sequence, enzymes, intermediates, products, and control mechanisms.
Learn the different stages of cellular respiration.
Know that the citric acid cycle involves the oxidation of 2-carbon units.
Be familiar with the function of the pyruvate dehydrogenase complex, its reaction types, general structure, and control mechanisms.
Understand how degradative reactions provide cycle intermediates.
Be familiar with role of the cycle in providing biosynthetic precursors.
Understand the role of anaplerotic reactions
Do NOT memorize specific enzyme mechanisms

3. Complete Oxidation to Molecular Oxygen
Glucose Note: 1 cal =4.184J
C6H12O6 + 6 O CO2 + 6 H2O  G° ’=-2823 kJ mole-1
Broken down into the half reactions:
C6H12O6 + 6 H2O 6CO2 + 24H e-
6 O H e H2O
Palmitic Acid
Palmitoyl-CoA + 23O Pi ADP CoA + 16CO H2O +131 ATP
Palmitic Acid + 23 O CO H2O  G°’= kJ mole-1
129 ADP + 129Pi ATP H2O  G°’= kJ mole-1
129 ATP is the next yield since 2 ATP are needed to form palmitoyl-CoA from palmitic acid. To Form 1 ATP  G°’= kJ mole-1 = 7.3 kcal mole-1
Citric Acid Cycle

4. Cellular Respiration Review
Citric Acid Cycle

5. Stage 1 of Cellular Respiration
Lehninger 2000 Fig 16.1a
Citric Acid Cycle

6. Stage 2 of Cellular Respiration
Lehninger 2000 Fig 16.1b
Citric Acid Cycle

7. Stage 3 of Cellular Respiration
Lehninger 2000 Fig 16.1c
Citric Acid Cycle

8. Overview of the Citric Acid Cycle
The central metabolic hub of the cell
The gateway to the aerobic metabolism for any molecule that can be converted into an acetyl group or a dicarboxylic acid.
Citric Acid Cycle

9. Coenzyme A Citric Acid Cycle
Berg, Tymoczko & Stryer, 2012 Chap. 17 p.497
Citric Acid Cycle

10. Acetyl Coenzyme A Acetyl CoA is the “fuel” for the citric acid cycle
Formed from the breakdown of glycogen, fats, and many amino acids.
A high energy compound G° = -31 kJ mol-1

11. Coenzyme A components
Lehninger 2000 Fig 16.3
Citric Acid Cycle

12. Mitochondrion Electron Micrograph
Citric Acid Cycle
Berg, Tymoczko & Stryer, 2012 Fig. 17.1

13. Citric Acid Cycle: Schematic Overview
acetyl group B
citrate
oxaloacetate
isocitrate
-ketoglutarate
succinate
Horton 2012 Fig. 13.5
Citric Acid Cycle
Berg, Tymoczko & Stryer, 2012 Fig. 17.2

14. Cellular Respiration Schematic
“The function of the citric acid cycle is the harvesting of high energy electrons from carbon fuels”.
Citric Acid Cycle
Berg, Tymoczko & Stryer, 2012 Fig. 17.3

15. Enzymes of the Citric Acid Cycle
Citrate synthase
Aconitase
Isocitrate dehydrogenase
-ketoglutarate dehydrogenase
Succinyl-CoA synthetase
Succinate dehydrogenase
Fumarase
Malate dehydrogenase
Citric Acid Cycle

16. Intermediates of the Citric Acid Cycle
oxaloacetate (4C)
citrate (6C)
cis-aconitate (6C)
isocitrate (6C)
-ketoglutarate (5C)
succinyl-CoA (4C)
succinate (4C)
fumarate (4C)
malate (4C)
Citric Acid Cycle

17. “Products” of the Citric Acid Cycle
Three (3) Hydride Ions (H-), that is six
(6) electrons are produced in the form of:
3 NADH (from isocitrate dehydrogenase,
-ketoglutarate dehydrogenase, & malate dehydrogenase)
1 FADH2 (from succinate dehydrogenase)
These electron carriers donate to electron transport which in turn drives oxidative phosphorylation to produce ATP
1 GTP (or ATP) (from succinyl CoA synthetase, a substrate-level phosphorylation)
2 CO2 (at isocitrate dehydrogenase & -ketoglutarate dehydrogenase)
Horton 2002 Fig12.6
Citric Acid Cycle

18. Citric Acid (Krebs) Cycle
Berg, Tymoczko & Stryer, 2012 Fig
Citric Acid Cycle

19. Citric Acid (Krebs) Cycle Rx List
Berg, Tymoczko & Stryer, 2012 Table 17.2
Citric Acid Cycle

20. Oxidation of Two Carbon Units [Citric Acid Cycle]

21. Glycolysis to the Citric Acid Cycle
Berg, Tymoczko & Stryer, Fig. 17.4

22. Pyruvate Dehydrogenase Complex Preparation to enter the Citric Acid Cycle

23. Pyruvate Dehydrogenase Reaction
Pyruvate + CoA + NAD+ acetyl CoA + CO2 + NADH
This is an irreversible reaction that links glycolysis and the citric acid cycle.
Citric Acid Cycle

24. Pyruvate Dehydrogenase Complex (E. Coli vs mammalian)
Citric Acid Cycle
Horton et al 2002, Table 12.1

25. Pyruvate Dehydrogenase Complex Schematic
PDH Azobacter vinelandii
core E3 E1
Berg, Tymoczko & Stryer, 2012 Fig. 17.7
Berg, Tymoczko & Stryer, 2001 Fig. 17.3
complete E2
Citric Acid Cycle
/ Pyruvate Dehydrogenase
Horton et al, 2002 Fig. 12.3
Voet, Voet, & Pratt 2012 Fig. 17.4

26. PDH Complex: Transacetylase (E2) Core
Citric Acid Cycle
/ Pyruvate Dehydrogenase
Berg, Tymoczko & Stryer, 2012 Fig. 17.8

27. Two of the cofactors in the Pyruvate Dehydrogenase Complex
Citric Acid Cycle
Berg, Tymoczko & Stryer, 2012 Chap 17 p.500

28. PDH Complex’s Three Basic Reaction Types
Citric Acid Cycle
Berg, Tymoczko & Stryer, 2012 Chap. 17 p. 500

29. Oxidative Decarboyxlation of Pyruvate by the PDH Complex
Citric Acid Cycle/ /
Pyruvate Dehydrogenase
Lehninger 2000 Fig 16.6

30. Pyruvate Dehydrogenase Complex Rx
Citric Acid Cycle
/ Pyruvate Dehydrogenase
Berg, Tymoczko & Stryer, 2012 Fig. 17.9

31. Formation of TPP Carbanion
TPP is the prosthetic group of pyruvate dehydrogenase.
Citric Acid Cycle
Berg, Tymoczko & Stryer, 2002 Chap 17 p. 500

32. Pyruvate Dehydrogenase Complex: Mechanisms
Citric Acid Cycle

33. Pyruvate Dehydrogenase Complex: Decarboxylation Reaction of E1
The charged TPP ring functions as an electron sink that acts to stabilize the transferred negative charge
Citric Acid Cycle
/ Pyruvate Dehydrogenase
Berg, Tymoczko & Stryer, 2012 Fig. 17.6

34. PDH Complex: Lipoamide Structure
Citric Acid Cycle
/ Pyruvate Dehydrogenase
Berg, Tymoczko & Stryer, 2002 Chap 17 p. 500

35. Structures and Interconversion of Lipoamide & Dihydrolipoamide
Voet, Voet & Pratt 2013 Figure 17.7

36. PDH Complex: Oxidation of the Hydroxyethyl Group and Transfer to Lipoamide
Carbanion
Catalyzed by pyruvate dehydrogenase component (E1).
Citric Acid Cycle
/ Pyruvate Dehydrogenase
Voet, Voet & Pratt 2013 Chap 17 p. 559

37. PDH Complex: Formation of Acetyl CoA by Transfer of Acetyl Group from Acetyllipoamide
Catalyzed by dihydrolipoyl transacetylase (E2).
Citric Acid Cycle
/ Pyruvate Dehydrogenase
Voet, Voet & Pratt 2013 Chap 17 p. 559

38. PDH Complex: Regeneration of Oxidized Form of Lipoamide by Dihydrolipoyl Dehydrogenase
Voet, Voet & Pratt 2013 Chap 17 p. 559
Summary of Two-step Process above
Citric Acid Cycle
/ Pyruvate Dehydrogenase
Berg, Tymoczko & Stryer, 2012 Chap 17

39. The Citric Acid Cycle
Citric Acid Cycle

40. Citric Acid Cycle Diagram: #1
Note that the acetyl group that enters the cycle does not give rise to the CO2 molecules given off in the decarboxylations in ONE TURN of the cycle.
Leheninger 2000 Fig 16.7

41. Citrate Synthase Structure
CLOSED
OPEN
Oxaloacetate binding induces the two domains to move toward each other in an 18 degree arc
This forms a binding site for acetyl CoA.
Berg, Tymoczko & Stryer Figure 17.10

42. Citric Acid Cycle: Condensation of Oxaloacetate & acetyl CoA
Citrate synthase
Citrate synthase
Reaction 1
G = kJ mol-1
Citric Acid Cycle
Berg, Tymoczko & Stryer, 2012 Chap 17 p.504

43. Citric Acid Cycle: Synthesis of Citryl CoA by Citrate Synthase
Berg, Tymoczko & Stryer, 2012 Fig
Citric Acid Cycle

44. Citric Acid Cycle Diagram: #2
Leheninger 2000 Fig 16.7

45. Citric Acid Cycle: Isomerization of Citrate by Aconitase
G = +8.4 kJ mol-1
G = -2.1 kJ mol-1
Reaction 2
The purpose of this reaction is to convert the citrate molecule to a secondary alcohol.
Voet, Voet & Pratt, 2013 Chap 17 p.563
Citric Acid Cycle

46. Citrate Binding to Aconitase’s Fe-S Complex
Citric Acid Cycle
Berg, Tymoczko & Stryer, 2012 Fig

47. Aconitase: Mechanism & Stereochemistry
double bond.
Voet & Voet Biochemistry 1995 Fig. 19.3
Citric Acid Cycle

48. Citric Acid Cycle Diagram: #3,4
Leheninger 2000 Fig 16.7

49. Citric Acid Cycle: Oxidative Decarboxylation of Isocitrate by Isocitrate Dehydrogenase
Do not dissociate from enzyme .
Reaction 3
G = -8.4 kJ mol-1
Voet, Voet, & Pratt Fig
Citric Acid Cycle

50. Citric Acid Cycle: Oxidative Decarboxylation of -ketoglutarate
-ketoglutarate dehydrogenase complex
Reaction 4
G = kJ mol-1
Berg, Tymoczko & Stryer, 2012 Chap 17 p.507
Citric Acid Cycle

51. Citric Acid Cycle Diagram: #5
Leheninger 2000 Fig 16.7

52. Citric Acid Cycle: Succinyl CoA Synthetase Reaction
G = -3.3 kJ mol-1
Berg, Tymoczko & Stryer, 2012 Chap 17 p.508
Citric Acid Cycle

53. Citric Acid Cycle: Succinyl CoA Synthetase Rx Mechanism
Berg, Tymoczko & Stryer, 2012 Fig
Citric Acid Cycle

54. Rx’s of Succinyl-CoA Synthetase
Citric Acid Cycle
Voet & Voet , & Pratt Fig

55. Citric Acid Cycle Diagram: #6,7
Leheninger 2000 Fig 16.7

56. Citric Acid Cycle: Succinate Dehydrogenase Rx
Reaction 6
G = 0 kJ mol-1
Voet, Voet & Pratt 2013 Chap 17 p. 567
Citric Acid Cycle

57. FAD vs NAD+ Reduction In general:
FAD functions biochemically to oxidize alkanes to alkenes. The oxidation of an alkane, e.g. succinate, to an alkene (fumarate) is sufficiently exergonic to reduce FAD to FADH2 but not to reduce NAD+.
NAD+ oxidizes alcohols to aldehydes or ketones. Alcohol oxidation can reduce NAD+ to NADH
Voet, Voet & Pratt 2013 p. 567; Voet & Voet 1996 p555
Citric Acid Cycle

58. Citric Acid Cycle: Hydration of Fumarate to Malate by Fumarase
Reaction 7
G = -3.8 kJ mol-1
Voet, Voet, & Pratt 2012 Chap p. 567
Citric Acid Cycle

59. Fumarate/Malate Stereochemistry
Berg, Tymoczko & Stryer, 2012 Chap. 17 p.510
Voet, Voet & Pratt 2006 Figure page 531
Citric Acid Cycle

60. Citric Acid Cycle: Oxidation of Malate to Oxaloacetate By Malate Dehydrogenase
Reaction 8
G = kJ mol-1
Voet, Voet, & Pratt 2013 Chap 17 p. 567
Citric Acid Cycle

61. Citric Acid Cycle Stoichiometry
Acetyl CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O
2 CO2 + 3 NADH + FADH2 + GTP + 2H+ + CoA
Citric Acid Cycle

62. Citric Acid (Krebs) Cycle
Acetyl group
Berg, Tymoczko & Stryer, 2012 Fig
Citric Acid Cycle

63. Citric Acid (Krebs) Cycle Reactions
Berg, Tymoczko & Stryer, 2012 Table 17.2
Citric Acid Cycle

64. Stoichiometry of ATP Formation Table
Citric Acid Cycle

65. Regulation of Entry Into and Metabolism Through the Citric Acid Cycle

66. Pathway from Glucose to Acetyl CoA
Citric Acid Cycle
Horton et la, , 2012 Fig
Voet, Voet & Pratt 2013 Chap 17 page 569

67. Regulation of the Pyruvate Dehydrogenase Complex
Berg, Tymoczko & Stryer, 2012 Fig
Berg, Tymoczko & Stryer, 2012 Fig a
Berg, Tymoczko & Stryer, 2002 Fig
Berg, Tymoczko & Stryer, 2012 Fig b
Citric Acid Cycle

68. Control of Metabolic Flux in the Cycle
Key Factors:
Substrate Availability
Inhibition by accumulating products
Allosteric feedback inhibition of enzymes that catalyze the cycle’s early reactions.
Enzyme Control Points:
Citrate synthase (Bacteria)
Isocitrate dehydrogenase
-ketoglutarate
Lehninger 2000, p 587
Citric Acid Cycle

69. Control of the Citric Acid Cycle
Berg, Tymoczko & Stryer, 2012 Fig
Citric Acid Cycle
Voet, Voet, & Pratt 2013 Fig

70. The Citric Acid Cycle and Biosynthetic Precursors

71. Anaplerotic Reactions Table (most common anaplerotic reactions)
Serve to replenish the citric acid cycle intermediates that are removed as biosynthetic precursors
Citric Acid Cycle

72. Degradative Pathways Generating Cycle Intermediates
Oxidation of odd chain fatty acids lead to the production of succinyl-CoA
Breakdown of the amino acids leucine, methionine and valine also lead to succinyl CoA production
Transamination and Deamination of amino acids leads to the production of -ketoglutarate and oxaloacetate.

73. Citric Acid Cycle: Roles in Biosynthesis
Berg, Tymoczko & Stryer, 2012 Fig
Citric Acid Cycle

74. The Citric Acid Cycle in Anabolism: Diagram

75. End of Lectures