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Lecture 4 - Citric Acid Cycle
Chem 454: Regulatory Mechanisms in Biochemistry
University of Wisconsin-Eau Claire
Introduction
The Citric Acid
Cycle is a
metabolic roundabout
It is the final
common
pathway for
oxidation of fuel
molecules
2
Introduction
Most material enters the Citric Acid Cycle as
Acetyl-CoA
The acetyl group
3
Introduction
For eukaryotes,
Citric Acid Cycle
located in the
mitochondrial
matrix
4
Introduction
Citric acid cycle is also an important source
of precursors
Two of the intermediates are only one step away
from an amino acid
One of the intermediates is used in the synthesis of
porphorins
Another is used in the synthesis of fatty acids and
sterols.
5
Introduction
6
Introduction
Citric acid cycle contains a series of
oxidation-reduction reactions
Carbon entering the cycle, leaves fully oxidized as
CO2.
“High energy” electrons leave the cycle with high
energy electron carriers as NADH and FADH2.
Very little ATP is made directly in the cycle.
No oxygen is used in the cycle.
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Introduction
8
Introduction
The “high energy” electrons are used
elsewhere to make ATP from ADP and Pi
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1. Oxidation of Two-Carbon Units
The citric acid cycle
oxidizes two carbon
units.
These enter the
cycle as Acetyl-CoA
Acetyl-CoA is
synthesized from
pyruvate or from fats
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1.1. Formation of Acetyl-CoA
Acetyl-CoA is formed from pyruvate by an
oxidative decarboxylation.
O
CH3
C
O
C
O
+
CoA-SH
Pyruvate
+
+
NAD
Dehydrogenase
Pyruvate
O
CH3
C
S
Acetyl-CoA
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CoA
+
O
C
O
+
NADH
1.2. Pyruvate Dehydrogenase Complex
Pyruvate Dehydrogenase
is a large multi-subunit
complex
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1.2. Pyruvate Dehydrogenase Complex
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1.2. Pyruvate Dehydrogenase Complex
Cofactors used
include
Thiamine
pyrophosphate
(TPP)
Lipoic Acid
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1.2. Pyruvate Dehydrogenase Complex
The pyruvate dehydrogenase reaction
involves three steps:
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1.2. Pyruvate Dehydrogenase Complex
(E1) - Pyruvated dehydrogenate component
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1.2. Pyruvate Dehydrogenase Complex
(E1) - Pyruvated dehydrogenate component
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1.2. Pyruvate Dehydrogenase Complex
(E2)-Dihydrolipoyl
transacetylase component
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1.2. Pyruvate Dehydrogenase Complex
(E2)-Dihydrolipoyl transacetylase component
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1.2. Pyruvate Dehydrogenase Complex
(E2)-Dihydrolipoyl transacetylase component
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1.2. Pyruvate Dehydrogenase Complex
(E3)-Dihydrolipoyl dehydrogenase component
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1.2. Pyruvate Dehydrogenase Complex
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1.2. Pyruvate Dehydrogenase Complex
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1.3. Citrate Synthase
First reaction of the citric acid cycle
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1.3. Citrate Synthase
The enzyme brings the two reactants into
juxtaposition
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1.4 Aconitase
Isomerizes citrate to isocitrate
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1.4 Aconitase
Aconitase contains a 4Fe-4S iron-sulfur
center
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1.5. Isocitrate Dehydrogenase
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1.6. α-Ketoglutarate Dehydrogenase
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1.6. α-Ketoglutarate Dehydrogenase
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1.7. Succinyl-CoA Synthetase
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1.7. Succinyl-CoA Synthetase
The mechanism involves a series of transfer
reactions
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1.8. Regeneration of Oxaloacetate
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1.9. Stoichiometry of Citric Acid Cycle
O
H3C
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C
S
CoA
+
3 NAD+
2 CO2
+
+
FAD
3 NADH
GDP
+
+
Pi
3 H+
+
+
2 H2O
FADH2
+
GTP
+
CoA-SH
1.9. Stoichiometry of Citric Acid Cycle
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1.9. Stoichiometry of Citric Acid Cycle
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Problem
What is the fate of the radioactive label
when each of the following compounds is
added to a cell extract containing the
enzymes and cofactors of the glycolytic
pathway, the citric acid cycle, and the
pyruvate dehydrogenase complex?
H3C
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O
O
O
O
C
C
C
C
COO
H3C
COO
H3C
COO
H3C
S
CoA
The Citric Acid Cycle
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Problem
In experiments carried out in 1941 to investigate the
citric acid cycle, oxaloacetate labeled with 14C in
the carboxyl carbon atom furthest from the keto
group was introduced to an active preparation of
mitochondria
Analysis of the α-ketoglutarate formed showed
that none of the radioactive label had been lost.
Decarboxylation of the α-ketoglutarate then
yielded succinate devoid of radioactivity. All the
label was in the released CO2. Why were the
early investigators of the citric acid cycle
surprised that all the label emerged in the CO2?
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Regulation of Citric Acid Cycle
The citric acid cycle
Final common pathway for oxidation of food
Also is a source of building blocks
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2.1. Regulation of Pyruvate Dehydrogenase
The pyruvate
dehydrogenase step
is irreversible in
animals
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2.1. Regulation of Pyruvate Dehydrogenase
Pyruvate Dehydrogenase is regulated both
allosterically and by reversible
phosphorylation
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2.2. Control Points in the Citric Acid Cycle
Citric acid cycle is
controlled at two
points
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3. Source or Biosynthetic Precursors
Citric acid cycle is also an important source
of precursors for biosynthetic reactions
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3.1. Replenishing the Intermediates
Pyruvate carboxylase reaction is used to
synthesize oxaloacetate from pyruvate
O
O
O
C
C
O
C
O
+ CO2 + ATP + H2O
CH3
O
C
CH2
C
Pyruvate
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O
O
Oxaloacetate
+ ATP + Pi + 2 H+
3.2. Disruption of Pyruvate Metabolism
Thiamine difficiency causes beriberi
3(AsO3 )
Arsenite
and mercury bind to
dithiols, such as dihydrolipoamide.
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The Glyoxylate Cycle
Some plants and
bacteria can live
off of acetate as a
fuel source.
These organisms
possess two
enzymes that
allow them to
carry out the
glyoxylate cycle:
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Problem
It is possible, with the use of the reactions
and enzymes discussed in this chapter, to
convert pyruvate into α-ketoglutarate
without depleting any of the citric acid
cycle components. Write a balanced
reaction scheme for this conversion,
showing cofactors and identifying the
required enzymes
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