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Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Chapter 20
The Tricarboxylic Acid Cycleto 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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
The TCA Cycleaka 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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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 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
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 CO2, 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 CyclePyruvate 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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
AconitaseIsomerization 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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
-Ketoglutarate DehydrogenaseA 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
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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Succinate DehydrogenaseAn 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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Malate DehydrogenaseAn 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!
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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)
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 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.
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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
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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
The Anaplerotic ReactionsThe "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.
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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’!
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Regulation of the TCA CycleAgain, 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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
The Glyoxylate CycleA 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 CO2-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
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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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