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 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



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





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





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



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!



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!













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









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!





-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





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







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









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







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!









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)



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.





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





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





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.







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’!





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







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





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







Documents

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