Deciphering the Tricarboxylic Acid Cycle: A Biochemical Journey

1. Chapter 20 The Tricarboxylic Acid Cycle to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777

2. Outline • 20.3 Bridging Step - Pyruvate Decarboxylase • 20.4 Entry - Citrate Synthase • 20.5 - 20.11 All the Other Steps • 20.13 Intermediates for Other Pathways • 20.14 Anaplerotic Reactions • 20.15 Regulation of the TCA Cycle • 20.16 The Glyoxylate Cycle

4. The TCA Cycle aka Citric Acid Cycle, Krebs Cycle • Pyruvate (actually acetate) from glycolysis is degraded to CO2 • Some ATP is produced • More NADH is made • NADH goes on to make more ATP in electron transport and oxidative phosphorylation

6. The Chemical Logic of TCAUnderstand this! • TCA seems like a complicated way to oxidize acetate units to CO2 • But normal ways to cleave C-C bonds and oxidize don't work for CO2: • cleavage between Cs  and  to a carbonyl • an -cleavage of an  -hydroxyketone

7. The Chemical Logic of TCAA better way to cleave acetate... • Better to condense acetate with oxaloacetate and carry out a  -cleavage - TCA combines this with oxidation to form CO2, regenerate oxaloacetate and capture all the energy as NADH and ATP!

8. Entry into the TCA Cycle Pyruvate dehydrogenase and citrate synthase • Pyruvate is oxidatively decarboxylated to form acetyl-CoA • Pyruvate dehydrogenase uses TPP, CoASH, lipoic acid, FAD and NAD+ • Citrate synthase is classic CoA chemistry! • Know both mechanisms • NADH & succinyl-CoA are allosteric inhibitors • Note (Table 20.1) that CS has large, neg G!

14. Aconitase Isomerization of Citrate to Isocitrate • Citrate is a poor substrate for oxidation • So aconitase isomerizes citrate to yield isocitrate which has a secondary-OH, which can be oxidized • Note the stereochemistry of the Rxn: aconitase removes the pro-R H of the pro-R arm of citrate! • Aconitase uses an iron-sulfur cluster - see Fig. 20.8

18. Isocitrate Dehydrogenase Oxidative decarboxylation of isocitrate to yield  -ketoglutarate • Classic NAD+ chemistry (hydride removal) followed by a decarboxylation • Isocitrate dehydrogenase is a link to the electron transport pathway because it makes NADH • Know the mechanism!

20.  -Ketoglutarate Dehydrogenase A second oxidative decarboxylation • This enzyme is nearly identical to pyruvate dehydrogenase - structurally and mechanistically • Five coenzymes used - TPP, CoASH, Lipoic acid, NAD+, FAD • You know the mechanism if you remember pyruvate dehydrogenase

22. Succinyl-CoA Synthetase A substrate-level phosphorylation • A nucleoside triphosphate is made • Its synthesis is driven by hydrolysis of a CoA ester • The mechanism (Figure 20.13) involves a phosphohistidine

25. Succinate Dehydrogenase An oxidation involving FAD • Mechanism involves hydride removal by FAD and a deprotonation • This enzyme is actually part of the electron transport pathway in the inner mitochondrial membrane • The electrons transferred from succinate to FAD (to form FADH2) are passed directly to ubiquinone (UQ) in the electron transport pathway

29. Fumarase Hydration across the double bond • trans-addition of the elements of water across the double bond • Possible mechanisms are shown in Figure 20.18 • The actual mechanism is not known for certain

32. Malate Dehydrogenase An NAD+-dependent oxidation • The carbon that gets oxidized is the one that received the-OH in the previous reaction • This reaction is energetically expensive • Go' = +30 kJ/mol • This and the previous two reactions form a reaction triad that we will see over and over!

36. TCA Cycle Summary One acetate through the cycle produces two CO2, one ATP, four reduced coenzymes • Make sure that you understand the equations on page 659 • A healthy exercise would be to try to derive these equations (or at least justify each term)

37. The Fate of Carbon in TCA Study Figure 20.21 carefully! • Carboxyl C of acetate turns to CO2 only in the second turn of the cycle (following entry of acetate) • Methyl C of acetate survives two cycles completely, but half of what's left exits the cycle on each turn after that.

41. Intermediates for BiosynthesisThe TCA cycle provides several of these •  -Ketoglutarate is transaminated to make glutamate, which can be used to make purine nucleotides, Arg and Pro • Succinyl-CoA can be used to make porphyrins • Fumarate and oxaloacetate can be used to make several amino acids and also pyrimidine nucleotides

43. Intermediates for BiosynthesisThe TCA cycle provides several of these • Note (Fig. 20.23) that mitochondrial citrate can be exported to be a cytoplasmic source of acetyl-CoA and oxaloacetate

45. The Anaplerotic Reactions The "filling up" reactions • PEP carboxylase - converts PEP to oxaloacetate • Pyruvate carboxylase - converts pyruvate to oxaloacetate • Malic enzyme converts pyruvate into malate • PEP carboxykinase - could have been an anaplerotic reaction, but it goes the wrong way! • CO2 binds weakly to the enzyme, but oxaloacetate binds tightly, so the reaction goes the wrong way.

48. The Reductive TCA Cycle • The TCA cycle running backward could assimilate CO2 • This may have been the first metabolic pathway • Energy to drive it? Maybe reaction of FeS with H2S to form FeS2 (iron pyrite) • iron pyrite, which was plentiful in ancient times, and which is an ancient version of ‘iron-sulfur clusters’!

50. Regulation of the TCA Cycle Again, 3 reactions are the key sites • Citrate synthase - ATP, NADH and succinyl-CoA inhibit • Isocitrate dehydrogenase - ATP inhibits, ADP and NAD+ activate •  -Ketoglutarate dehydrogenase - NADH and succinyl-CoA inhibit, AMP activates • Also note pyruvate dehydrogenase: ATP, NADH, acetyl-CoA inhibit, NAD+, CoA activate