Chem 454: Regulatory Mechanisms in BiochemistryUniversity of Wisconsin-Eau Claire

Lecture 4 - Citric Acid Cycle

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Introduction

The Citric Acid Cycle is a metabolic round-aboutIt is the final common pathway for oxidation of fuel molecules

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Most material enters the Citric Acid Cycle as Acetyl-CoA

Introduction

The acetyl group

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For eukaryotes, Citric Acid Cycle located in the mitochondrial matrix

Introduction

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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.

Introduction

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Introduction

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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.

Introduction

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Introduction

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The “high energy” electrons are used elsewhere to make ATP from ADP and Pi

Introduction

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The citric acid cycle oxidizes two carbon units.

These enter the cycle as Acetyl-CoA

Acetyl-CoA is synthesized from pyruvate or from fats

1. Oxidation of Two-Carbon Units

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Acetyl-CoA is formed from pyruvate by an oxidative decarboxylation.

1.1. Formation of Acetyl-CoA

Pyruvate+ CoA-SH + NAD+C C

OO

OCH3

Acetyl-CoACO

CH3 S CoA + + NADHO C O

PyruvateDehydrogenase

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Pyruvate Dehydrogenase is a large multi-subunit complex

1.2. Pyruvate Dehydrogenase Complex

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1.2. Pyruvate Dehydrogenase Complex

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Cofactors used include

Thiamine pyrophosphate (TPP)

Lipoic Acid

1.2. Pyruvate Dehydrogenase Complex

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The pyruvate dehydrogenase reaction involves three steps:

1.2. Pyruvate Dehydrogenase Complex

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(E1) - Pyruvated dehydrogenate component

1.2. Pyruvate Dehydrogenase Complex

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(E1) - Pyruvated dehydrogenate component

1.2. Pyruvate Dehydrogenase Complex

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(E2)-Dihydrolipoyl transacetylase component

1.2. Pyruvate Dehydrogenase Complex

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(E2)-Dihydrolipoyl transacetylase component

1.2. Pyruvate Dehydrogenase Complex

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(E2)-Dihydrolipoyl transacetylase component

1.2. Pyruvate Dehydrogenase Complex

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(E3)-Dihydrolipoyl dehydrogenase component

1.2. Pyruvate Dehydrogenase Complex

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1.2. Pyruvate Dehydrogenase Complex

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1.2. Pyruvate Dehydrogenase Complex

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First reaction of the citric acid cycle

1.3. Citrate Synthase

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The enzyme brings the two reactants into juxtaposition

1.3. Citrate Synthase

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Isomerizes citrate to isocitrate

1.4 Aconitase

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Aconitase contains a 4Fe-4S iron-sulfur center

1.4 Aconitase

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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

H3C CO

S CoA + 3 NAD+ + FAD GDP Pi + 2 H2O

2 CO2 + 3 NADH + FADH2 + GTP + CoA-SH

+

3 H++ 34

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1.9. Stoichiometry of Citric Acid Cycle

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1.9. Stoichiometry of Citric Acid Cycle

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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?

Problem

H3CCO

COO H3CCO

COO H3CCO

COO H3CCO

S CoA

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The Citric Acid Cycle

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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?

Problem

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The citric acid cycle

Final common pathway for oxidation of food

Also is a source of building blocks

Regulation of Citric Acid Cycle

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The pyruvate dehydrogenase step is irreversible in animals

2.1. Regulation of Pyruvate Dehydrogenase

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Pyruvate Dehydrogenase is regulated both allosterically and by reversible phosphorylation

2.1. Regulation of Pyruvate Dehydrogenase

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Citric acid cycle is controlled at two points

2.2. Control Points in the Citric Acid Cycle

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Citric acid cycle is also an important source of precursors for biosynthetic reactions

3. Source or Biosynthetic Precursors

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Pyruvate carboxylase reaction is used to synthesize oxaloacetate from pyruvate

3.1. Replenishing the Intermediates

CC

O O

OCH3

+ CO2 + ATP + H2OCC

O O

OCH2

+

CO O

ATP + Pi + 2 H+

Pyruvate Oxaloacetate

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Thiamine difficiency causes beriberi

Arsenite (AsO33-) and mercury bind to

dithiols, such as dihydrolipoamide.

3.2. Disruption of Pyruvate Metabolism

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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:

The Glyoxylate Cycle

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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

Problem

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