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Berg Tymoczko Stryer

Biochemistry
Sixth Edition
Chapter 17:
Th Cit i A id C l The Citric Acid Cycle
Copyright 2007 by W. H. Freeman and Company
The Citric Acid Cycle
Glycolysis produces just
2 ATP molecules
Aerobic metabolism of
glucose CO
2
gives
more ATP
Main part called the citric
acid cycle
Tricarboxylic acid (TCA)
cycle cycle
Krebs cycle

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The Citric Acid Cycle
Pyruvate must be converted to acetyl-CoA
Pyruvate + coenzyme A + NAD
+
acetyl-CoA +
CO + NADH CO
2
+ NADH
Acetyl-CoA then enters the citric acid cycle,
which occurs inside mitochondria
The Citric Acid Cycle
Citric acid cycle the
metabolic hub of the
cell
6 carbon
tricarboxylic
acid
cell
Fuels aerobically
oxidized
A source of
precursors for amino
acids nucleotide acids, nucleotide
bases, porphyrin

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The Citric Acid Cycle
How does the citric acid cycle connect to other
metabolic pathways?
The Citric Acid Cycle
Pyruvate + coenzyme A + NAD
+
acetyl-CoA +
CO
2
+ NADH
Pyruvate transported through membrane protein Pyruvate transported through membrane protein
into mitochondria
Pyruvate dehydrogenase complex catalyzes this
irreversible reaction
Complex of 3 enzymes
Member of a large family with masses from 4 million to Member of a large family, with masses from 4 million to
10 million daltons

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The Citric Acid Cycle
The Citric Acid Cycle
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Mechanism of Pyruvate Acetyl CoA
Pyruvate + CoA + NAD
+
Acetyl CoA + CO
2
+ NADH + H
+
Requires 5 coenzymes
Catalytic cofactors: thiamine pyrophosphate (TPP), lipoic acid, and
FAD FAD
Stoichiometric cofactors: CoA and NAD+

Mechanism of Pyruvate Acetyl CoA

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Mechanism of Pyruvate Acetyl CoA
Decarboxylation
Catalyzed by E
1
of pyruvate dehydrogenase
complex complex
Mechanism of Pyruvate Acetyl CoA
Oxidation
Catalyzed by the pyruvate
dehydrogenase component of the dehydrogenase component of the
complex (E
1
)

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Mechanism of Pyruvate Acetyl CoA
Transfer to CoA
Catalyzed by dihydrolipoyl transacetylase (E
2
)
Thioester bond remains in product Thioester bond remains in product

Mechanism of Pyruvate Acetyl CoA

Step 4: Dihydrolipoamide oxidized to lipoamide
Catalyzed by dihydrolipoyl dehydrogenase (E
3
)
2 e transferred to FAD then to NAD
+
2 e- transferred to FAD then to NAD
+

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Mechanism of Pyruvate Acetyl CoA
Complex structure of the complex
12 E
3
() N-terminus
24 E
1
(
2

2
)
8 E
2
(
3
)
Mechanism of Pyruvate Acetyl CoA
Advantages of a compact multienzyme complex
Reactions more efficient because reactants and
enzymes so close to each other increases overall enzymes so close to each other, increases overall
rate and minimizes side reactions
Lipoamide swings to pyruvate dehydrogenase to
accept acetyl group
Swings to transacetylase to transfer it to CoA-SH
Swings to dihydrolipoyl dehydrogenase to regenerate
sulfhydryl groups

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Reactions of the Citric Acid Cycle
1, Formation of citrate, a condensation reaction
Catalyzed by citrate synthase

Reactions of the Citric Acid Cycle

Mechanism of citrate synthase, how does it
prevent hydrolysis of acetyl CoA?
Large conformational changes during catalysis Large conformational changes during catalysis

Oxaloacetat
e
Acetyl CoA CoA Citrate
Enzym
e
Enzyme
Condensation

Reaction
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Reactions of the Citric Acid Cycle
Acetyl CoA transformed
to enol intermediate
Citryl CoA causes
conformational
changes that close
active site active site
Reactions of the Citric Acid Cycle
2, Isomerization of citrate to isocitrate

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Reactions of the Citric Acid Cycle
Aconitase in a class called iron-sulfur proteins

4 Fe atoms complexed to 4 sulfides and 3

cysteine S, one Fe binds to citrate through COO-
& OH groups
Reactions of the Citric Acid Cycle
Fluoracetatyl-CoA also a substrate for citrate
synthase
Fluoracetate found in leaves of some poisonous Fluoracetate found in leaves of some poisonous
plants
Fluorocitrate inhibits aconitase (enzyme in next
rxn of citric acid cycle)
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Reactions of the Citric Acid Cycle
3, 1
st
oxidation, formation of -ketoglutarate and
CO
2

Reactions of the Citric Acid Cycle

4, 2
nd
oxidation, formation of succinyl-CoA and
CO
2
Another oxidative decarboxylation catalyzed by Another oxidative decarboxylation, catalyzed by
the -ketoglutarate dehydrogenase complex

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Reactions of the Citric Acid Cycle
5, Formation of succinate
Catalyzed by succinyl CoA synthetase

Reactions of the Citric Acid Cycle

6, Formation of fumarate, an FAD-linked
oxidation
Catalyzed by succinate dehydrogenase an Catalyzed by succinate dehydrogenase, an
integral protein of the mitochondrial membrane,
also is directly associated with the electron-
transport chain
Because FAD covalently bound, transfer e- to Fe-
S clusters of the protein, then to electron transport
E E
S clusters of the protein, then to electron transport
chain

E- E-
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Reactions of the Citric Acid Cycle
7, Formation of L-malate by hydration

Reactions of the Citric Acid Cycle

8, The final oxidation, regeneration of
oxaloacetate
Catalyzed by malate dehydrogenase Catalyzed by malate dehydrogenase
2.5 ATP for each NADH

G
o
= + 29.7 kJ/mol
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Reactions of the Citric Acid Cycle
Net of steps 6-8

Summary of Reactions
Pyruvate dehydrogenase complex in conjunction
with the citric acid cycle yields

Pyruvate dehydrogenase complex:

Pyruvate + CoA-SH + NAD+ Acetyl-CoA +
NADH + CO
2
+ H
+
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Summary of Reactions
Citric acid cycle
Acetyl-CoA + 3 NAD
+
+ FAD + GDP + P
i
+ 2 H
2
O
2 CO
2
+ COA-SH + 3 NADH + 3 H
+
+ FADH
2
+ 2 CO
2
+ COA SH + 3 NADH + 3 H + FADH
2
+
GTP
Overall reaction
Pyruvate + 4 NAD
+
+ FAD + GDP + P
i
+ 2 H
2
O
3 CO
2
+ 4 NADH + FADH
2
+ GTP + 4 H
+
E t l ATP d ti t Eventual ATP production per pyruvate:
4 NADH 10 ATP
1 FADH
2
1.5 ATP
1 GTP 1 ATP
Sum: 12.5 ATP per pyruvate (25 per glucose)
Summary of Reactions
Interesting points
Enzymes of the citric acid cycle may be physically
associated with each other leading products to associated with each other, leading products to
pass directly from one to the other in a process
called substrate channeling
Citric acid cycle strictly aerobic because O
2
required to regenerate NAD
+
and FAD in the
mitochondrion toc o d o
Net pyruvate acetyl CoA has G
o
= -33.4
kJ/mol

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Metabolic Control
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Metabolic Control
Entry into cycle & rate of cycle
tightly controlled
Pyruvate acetyl CoA Pyruvate acetyl CoA
irreversible in animals
C oxidized to CO
2
by TCA cycle
Incorporated into lipids
Pyruvate dehydrogenase (PDH)
complex inhibited by products

Metabolic Control
PDH complex activated by ADP and pyruvate

ADP & pyruvate inhibit the kinase that turns off ADP & pyruvate inhibit the kinase that turns off
PDH
Both the kinase and phosphatase are associated
with the PDH complex
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Metabolic Control
How is the phosphatase activated?
Recall the -adrenergic receptor is stimulated by
epinephrine leads to release of Ca
2+
into epinephrine, leads to release of Ca into
cytoplasm and stimulates muscle contraction

Metabolic Control
Citric acid cycle controlled at 3 points, rxns of
Citrate synthase, isocitrate dehydrogenase, & the
-ketoglutarate dehydrogenase complex g y g p
Citrate synthase
Inhibited by
Activated by
Isocitrate dehydrogenase
Activated by Activated by
Inhibited by
The -ketoglutarate dehydrogenase complex
Inhibited by
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Metabolic Control
Cells in a resting Cells in a highly active
Relationship between metabolic state of a cell and the ATP/ADP
and NADH/NAD
+
ratios
Cells in a resting
metabolic state
Need and use little
energy
High ATP, low ADP
l l i l hi h
Cells in a highly active
metabolic state
Need and use more
energy than resting cells
Low ATP, high ADP
levels imply low
ATP/ADP ratio
levels imply high
ATP/ADP ratio
High NADH, low NAD
+
levels imply high
NADH/NAD
+
ratio
ATP/ADP ratio
Low NADH, high NAD
+
levels imply low
NADH/NAD
+
ratio
Metabolic Control
Inhibition of isocitrate dehydrogenase leads to
buildup of citrate
Citrate signals glycolysis to stop Citrate signals glycolysis to stop
Can be a source of acetyl CoA for fatty acid
synthesis
Inhibition of -ketoglutarate dehydrogenase
leads to buildup of -ketoglutarate
Used as precursor for synthesis of many amino
acids and purine baes
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Metabolic Control
TCA Cycle & Anabolism
Supply of cycle components need to be
replenished to keep cycle operating as they are
used for synthesis used for synthesis
Anaplerotic reaction reaction that replenishes a
citric acid cycle intermediate
[Oxaloacetate] must allow acetyl-CoA to enter
cycle
In mammals Pyruvate + CO + ATP + H O In mammals, Pyruvate + CO
2
+ ATP + H
2
O
oxaloacetate + ADP + P
i
+ 2 H
+

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TCA Cycle & Anabolism
Beriberi
Beriberi a disorder caused by a lack of
thiamine (vitamin B
1
) in the diet, results in weight
loss, pain, emotional disturbance, weakness, loss, pain, emotional disturbance, weakness,
irregular heart rate
Rare except in the Far East where rice is major
food
Rice has a low content of thiamine
O i ll l h li ill b l i h d Occasionally alcoholics will be malnourished
enough to suffer beriberi
What is the biochemistry of this?
Thiamine is precursor to thiamine pyrophosphate
(TPP), a coenzyme of pyruvate dehydrogenase,
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Beriberi
Beriberi
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Glyoxylate Cycle
Plants and bacteria
can synthesize
carbohydrates from carbohydrates from
acetyl-CoA
Similar to TCA
cycle, but
decarboxylations
bypassed & 2
Unique
reactions of
glyoxylate
cycle
bypassed & 2
acetyl-CoA
molecules enter per
cycle
Lets them grow on
acetate
Carbohydrate
s
Summary
Pyruvate dehydrogenase complex links
glycolysis to the citric acid cycle
TCA cycle starts with condensation of 4C + 2C TCA cycle starts with condensation of 4C + 2C
molecule, 4C molecule regenerated
12.5 ATP/pyruvate from TCA cycle & PDH
reaction
TCA cycle tightly controlled
Control closely tied to energy status of cell
TCA cycle gives provide synthetic precursors
Glyoxylate cycle lets plants & bacteria
synthesize carbohydrates from acetyl-CoA