Lecture 4 - Citric Acid Cycle
Chem 454: Regulatory Mechanisms in Biochemistry University of Wisconsin-Eau Claire
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Introduction
The Citric Acid Cycle is a metabolic round- about It is the final common pathway for oxidation of fuel molecules
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Introduction
Most material enters the Citric Acid Cycle as Acetyl-CoA
The acetyl group
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Introduction
For eukaryotes, Citric Acid Cycle located in the mitochondrial matrix
4 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.
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Introduction
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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 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 O + Pyruvate CH3 C C O + CoA-SH + NAD Dehydrogenase Pyruvate O
CH3 C S CoA + O C O + NADH Acetyl-CoA
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1.2. Pyruvate Dehydrogenase Complex
Pyruvate Dehydrogenase is a large multi-subunit complex
12 12 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
16 16 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
20 20 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
24 24 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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28 28 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
32 32 1.8. Regeneration of Oxaloacetate
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1.9. Stoichiometry of Citric Acid Cycle
O + FAD H3C C S CoA + 3 NAD + GDP + Pi + 2 H2O
2 CO + + + + + + CoA-SH 34 2 3 NADH 3 H FADH2 GTP 34
1.9. Stoichiometry of Citric Acid Cycle
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1.9. Stoichiometry of Citric Acid Cycle
36 36 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?
O O O O
C C C C
H3C COO H3C COO H3C COO H3C S CoA
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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? 39 39
Regulation of Citric Acid Cycle
The citric acid cycle
Final common pathway for oxidation of food
Also is a source of building blocks
40 40 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
44 44 3.1. Replenishing the Intermediates
Pyruvate carboxylase reaction is used to synthesize oxaloacetate from pyruvate
O O O O C C + O C + CO2 + ATP + H2O O C + ATP + Pi + 2 H
CH3 CH2 C O O Pyruvate Oxaloacetate
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3.2. Disruption of Pyruvate Metabolism
Thiamine difficiency causes beriberi 3- Arsenite (AsO3 ) 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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