Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 20 The Tricarboxylic Acid Cycle to accompany Biochemistry, 2/e.

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The TCA Cycle aka Citric Acid Cycle, Krebs Cycle Pyruvate (actually acetate) from glycolysis is degraded to CO 2 Some ATP is produced More NADH is made NADH goes on to make more ATP in electron transport and oxidative phosphorylation

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The Chemical Logic of TCA Understand this! TCA seems like a complicated way to oxidize acetate units to CO 2 But normal ways to cleave C-C bonds and oxidize don't work for CO 2 : –cleavage between Cs  and  to a carbonyl –an  -cleavage of an  -hydroxyketone

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The Chemical Logic of TCA A better way to cleave acetate... Better to condense acetate with oxaloacetate and carry out a  - cleavage - TCA combines this with oxidation to form CO 2, regenerate oxaloacetate and capture all the energy as NADH and ATP!

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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!

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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!

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company  -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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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 FADH 2 ) are passed directly to ubiquinone (UQ) in the electron transport pathway

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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  G o ' = +30 kJ/mol This and the previous two reactions form a reaction triad that we will see over and over!

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company TCA Cycle Summary One acetate through the cycle produces two CO 2, 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)

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The Fate of Carbon in TCA Study Figure 20.21 carefully! Carboxyl C of acetate turns to CO 2 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.

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Intermediates for Biosynthesis The 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Intermediates for Biosynthesis The 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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! CO 2 binds weakly to the enzyme, but oxaloacetate binds tightly, so the reaction goes the wrong way.

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The Reductive TCA Cycle The TCA cycle running backward could assimilate CO 2 This may have been the first metabolic pathway Energy to drive it? Maybe reaction of FeS with H 2 S to form FeS 2 (iron pyrite) iron pyrite, which was plentiful in ancient times, and which is an ancient version of ‘iron-sulfur clusters’!

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The Glyoxylate Cycle A variant of TCA for plants and bacteria Acetate-based growth - net synthesis of carbohydrates and other intermediates from acetate - is not possible with TCA Glyoxylate cycle offers a solution for plants and some bacteria and algae The CO 2 -evolving steps are bypassed and an extra acetate is utilized Isocitrate lyase and malate synthase are the short-circuiting enzymes

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Glyoxylate Cycle II Isocitrate lyase produces glyoxylate and succinate Malate synthase does a Claisen condensation of acetyl-CoA and the aldehyde group of glyoxylate - classic CoA chemistry! The glyoxylate cycle helps plants grow in the dark! Glyoxysomes borrow three reactions from mitochondria: succinate to oxaloacetate

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 20 The Tricarboxylic Acid Cycle to accompany Biochemistry, 2/e.

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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 20 The Tricarboxylic Acid Cycle to accompany Biochemistry, 2/e.

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