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Biochemistry 2/e - Garrett & Grisham
Chapter 20
The Tricarboxylic Acid Cycle
to accompany
Biochemistry, 2/e
by
Reginald Garrett and Charles Grisham
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Biochemistry 2/e - Garrett & Grisham
Outline
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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
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
The TCA Cycle
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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
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Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
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
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Biochemistry 2/e - Garrett & Grisham
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!
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Biochemistry 2/e - Garrett & Grisham
Entry into the TCA Cycle
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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!
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Biochemistry 2/e - Garrett & Grisham
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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
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Biochemistry 2/e - Garrett & Grisham
Aconitase
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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
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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!
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
 -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
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Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
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
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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
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Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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!
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
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)
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Biochemistry 2/e - Garrett & Grisham
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.
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
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
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Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
The Anaplerotic Reactions
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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.
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
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’!
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Regulation of the TCA Cycle
Again, 3 reactions are the key sites
• Citrate synthase - ATP, NADH and succinylCoA 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
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
The Glyoxylate Cycle
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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 CO2-evolving steps are bypassed and
an extra acetate is utilized
Isocitrate lyase and malate synthase are the
short-circuiting enzymes
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Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
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
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company