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

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

Complete Oxidation to Molecular Oxygen Glucose Note: 1 cal =4.184J C6H12O6 + 6 O2 6 CO2 + 6 H2O  G° ’=-2823 kJ mole-1 Broken down into the half reactions: C6H12O6 + 6 H2O 6CO2 + 24H+ + 24 e- 6 O2 + + 24H+ + 24 e- 12 H2O Palmitic Acid Palmitoyl-CoA + 23O2 + 131 Pi + 131 ADP CoA + 16CO2 + 146 H2O +131 ATP Palmitic Acid + 23 O2 16 CO2 + 16 H2O  G°’= -9790.5 kJ mole-1 129 ADP + 129Pi 129 ATP + 129 H2O  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

Cellular Respiration Review Citric Acid Cycle

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

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

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

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

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

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

Coenzyme A components Lehninger 2000 Fig 16.3 Citric Acid Cycle

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

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

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

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

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

“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

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

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

Oxidation of Two Carbon Units [Citric Acid Cycle]

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

Pyruvate Dehydrogenase Complex Preparation to enter the Citric Acid Cycle

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

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

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

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

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

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

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

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

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

Pyruvate Dehydrogenase Complex: Mechanisms Citric Acid Cycle

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

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

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

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

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

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

The Citric Acid Cycle Citric Acid Cycle

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

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

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

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

Citric Acid Cycle Diagram: #2 Leheninger 2000 Fig 16.7

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

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

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

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

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. 17.11 Citric Acid Cycle

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

Citric Acid Cycle Diagram: #5 Leheninger 2000 Fig 16.7

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

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

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

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

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

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

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

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

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

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

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

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

Stoichiometry of ATP Formation Table Citric Acid Cycle

Regulation of Entry Into and Metabolism Through the Citric Acid Cycle

Pathway from Glucose to Acetyl CoA Citric Acid Cycle Horton et la, , 2012 Fig. 13.11 Voet, Voet & Pratt 2013 Chap 17 page 569

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

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

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

The Citric Acid Cycle and Biosynthetic Precursors

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

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.

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

The Citric Acid Cycle in Anabolism: Diagram

End of Lectures