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

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Presentation on theme: "CITRIC ACID CYCLE Student Edition 11/8/13 version Pharm. 304 Biochemistry Fall 2014 Dr. Brad Chazotte 213 Maddox Hall Web Site:"— Presentation transcript:

1 CITRIC ACID CYCLE Student Edition 11/8/13 version Pharm. 304 Biochemistry Fall 2014 Dr. Brad Chazotte 213 Maddox Hall chazotte@campbell.edu Web Site: http://www.campbell.edu/faculty/chazotte http://www.campbell.edu/faculty/chazotte Original material only ©2002-14 B. Chazotte

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 C 6 H 12 O 6 + 6 O 2 6 CO 2 + 6 H 2 O  G° ’=-2823 kJ mole -1 Broken down into the half reactions: C 6 H 12 O 6 + 6 H 2 O 6CO 2 + 24H + + 24 e- 6 O 2 + + 24H + + 24 e - 12 H 2 O Palmitic Acid Palmitoyl-CoA + 23O 2 + 131 P i + 131 ADP CoA + 16CO 2 + 146 H 2 O +131 ATP Palmitic Acid + 23 O 2 16 CO 2 + 16 H 2 O  G°’= -9790.5 kJ mole -1 129 ADP + 129P i 129 ATP + 129 H 2 O  G°’= +3941 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°’= +30.54 kJ mole -1 = 7.3 kcal mole -1 Citric Acid Cycle

4 Cellular Respiration Review Citric Acid Cycle

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

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

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

8 Overview of the Citric Acid Cycle 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.

9 Berg, Tymoczko & Stryer, 2012 Chap. 17 p.497 Coenzyme A 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 Lehninger 2000 Fig 16.3 Coenzyme A components Citric Acid Cycle

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

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

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

15 Enzymes of the Citric Acid Cycle 1.Citrate synthase 2.Aconitase 3.Isocitrate dehydrogenase 4.  -ketoglutarate dehydrogenase 5.Succinyl-CoA synthetase 6.Succinate dehydrogenase 7.Fumarase 8.Malate dehydrogenase Citric Acid Cycle

16 Intermediates of the Citric Acid Cycle 1.oxaloacetate (4C) 2.citrate (6C) 3.cis-aconitate (6C) 4.isocitrate (6C) 5.  -ketoglutarate (5C) 6.succinyl-CoA (4C) 7.succinate(4C) 8.fumarate(4C) 9.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 FADH 2 (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 CO 2 (at isocitrate dehydrogenase &  -ketoglutarate dehydrogenase) Citric Acid Cycle Horton 2002 Fig12.6

18 Berg, Tymoczko & Stryer, 2012 Fig. 17.15 Citric Acid (Krebs) Cycle Citric Acid Cycle

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

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

21 Berg, Tymoczko & Stryer, 2012 Fig. 17.4 Glycolysis to the Citric Acid Cycle Citric Acid Cycle

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

23 Pyruvate Dehydrogenase Reaction Pyruvate + CoA + NAD + acetyl CoA + CO 2 + NADH This is an irreversible reaction that links glycolysis and the citric acid cycle. Citric Acid Cycle

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

25 Berg, Tymoczko & Stryer, 2012 Fig. 17.7 Pyruvate Dehydrogenase Complex Schematic Citric Acid Cycle / Pyruvate Dehydrogenase Horton et al, 2002 Fig. 12.3 E1E1 E2E2 E3E3 PDH Azobacter vinelandii core complete Berg, Tymoczko & Stryer, 2001 Fig. 17.3 Voet, Voet, & Pratt 2012 Fig. 17.4

26 Berg, Tymoczko & Stryer, 2012 Fig. 17.8 PDH Complex: Transacetylase (E 2 ) Core Citric Acid Cycle / Pyruvate Dehydrogenase

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

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

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

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

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

32 Pyruvate Dehydrogenase Complex: Mechanisms Citric Acid Cycle

33 Berg, Tymoczko & Stryer, 2012 Fig. 17.6 Pyruvate Dehydrogenase Complex: Decarboxylation Reaction of E 1 Citric Acid Cycle / Pyruvate Dehydrogenase The charged TPP ring functions as an electron sink that acts to stabilize the transferred negative charge

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

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

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

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

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

39 The Citric Acid Cycle Citric Acid Cycle

40 Leheninger 2000 Fig 16.7 Citric Acid Cycle Diagram: #1 1 Note that the acetyl group that enters the cycle does not give rise to the CO 2 molecules given off in the decarboxylations in ONE TURN of the cycle.

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

42 Berg, Tymoczko & Stryer, 2012 Chap 17 p.504 Citric Acid Cycle: Condensation of Oxaloacetate & acetyl CoA Citric Acid Cycle Citrate synthase  G  = -31.4 kJ mol -1 Reaction 1

43 Berg, Tymoczko & Stryer, 2012 Fig. 17.11 Citric Acid Cycle: Synthesis of Citryl CoA by Citrate Synthase Citric Acid Cycle

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

45 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: Isomerization of Citrate by Aconitase Citric Acid Cycle Aconitase  G  = +8.4 kJ mol -1  G  = -2.1 kJ mol -1 Reaction 2

46 Berg, Tymoczko & Stryer, 2012 Fig. 17.12 Citrate Binding to Aconitase’s Fe-S Complex Citric Acid Cycle

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

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

49 Voet, Voet, & Pratt Fig. 17.11 Citric Acid Cycle: Oxidative Decarboxylation of Isocitrate by Isocitrate Dehydrogenase Citric Acid Cycle Isocitrate dehydrogenase  G  = -8.4 kJ mol -1 Reaction 3 Do not dissociate from enzyme

50 Berg, Tymoczko & Stryer, 2012 Chap 17 p.507 Citric Acid Cycle: Oxidative Decarboxylation of  -ketoglutarate Citric Acid Cycle  -ketoglutarate dehydrogenase complex  G  = -30.1 kJ mol -1 Reaction 4 

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

52 Berg, Tymoczko & Stryer, 2012 Chap 17 p.508 Citric Acid Cycle: Succinyl CoA Synthetase Reaction Citric Acid Cycle Succinyl CoA Synthetase  G  = -3.3 kJ mol -1 Reaction 5

53 Berg, Tymoczko & Stryer, 2012 Fig. 17.13 Citric Acid Cycle: Succinyl CoA Synthetase R x Mechanism Citric Acid Cycle

54 Rx’s of Succinyl-CoA Synthetase Voet & Voet, & Pratt 2013 Fig. 17.12 Citric Acid Cycle

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

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

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 FADH 2 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 Voet, Voet, & Pratt 2012 Chap.. 17 p. 567 Citric Acid Cycle: Hydration of Fumarate to Malate by Fumarase Citric Acid Cycle Fumarase  G  = -3.8 kJ mol -1 Reaction 7 Fumarase

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

60 Voet, Voet, & Pratt 2013 Chap 17 p. 567 Citric Acid Cycle: Oxidation of Malate to Oxaloacetate By Malate Dehydrogenase Citric Acid Cycle Malate dehydrogenase  G  = +29.7 kJ mol -1 Reaction 8

61 Citric Acid Cycle Stoichiometry Citric Acid Cycle Acetyl CoA + 3 NAD + + FAD + GDP + P i + 2 H 2 O 2 CO 2 + 3 NADH + FADH 2 + GTP + 2H + + CoA

62 Berg, Tymoczko & Stryer, 2012 Fig. 17.15 Citric Acid (Krebs) Cycle Citric Acid Cycle

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

64 Stoichiometry of ATP Formation Table

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. 13.11 Voet, Voet & Pratt 2013 Chap 17 page 569

67 Berg, Tymoczko & Stryer, 2002 Fig. 17.17 Regulation of the Pyruvate Dehydrogenase Complex Citric Acid Cycle Berg, Tymoczko & Stryer, 2012 Fig. 17.18a Berg, Tymoczko & Stryer, 2012 Fig. 17.18b Berg, Tymoczko & Stryer, 2012 Fig. 17.17

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. Lehninger 2000, p 587 Citric Acid Cycle Enzyme Control Points: Citrate synthase (Bacteria) Isocitrate dehydrogenase  -ketoglutarate

69 Berg, Tymoczko & Stryer, 2012 Fig. 17.19 Control of the Citric Acid Cycle Citric Acid Cycle Voet, Voet, & Pratt 2013 Fig. 17.16

70 The Citric Acid Cycle and Biosynthetic Precursors Citric Acid Cycle

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

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 Berg, Tymoczko & Stryer, 2012 Fig. 17.20 Citric Acid Cycle: Roles in Biosynthesis Citric Acid Cycle

74 The Citric Acid Cycle in Anabolism: Diagram

75 End of Lectures


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