The Citric Acid Cycle

Michael Borenstein, Ph.D.

Cellular respiration and energy production

- aerobic phase of catabolism comprising processes by which cells consume O2 and produce CO2 and energy = cellular respiration comprises three stages: 1. oxidation of organic fuels to acetylCoA

2.

conversion of acetylCoA to CO2 and reduced forms of electron carriers NADH, FADH2

3.

oxidation of electron carriers with O2 (final electron acceptor) with release of energy, converted to ATP (oxidative phosphorylation)
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Cellular respiration and energy production

- carried out in mitochondria:

Oxidation of pyruvate to acetylCoA

- oxidative decarboxylation catalyzed by a cluster of enzymes and their cofactors = pyruvate dehydrogenase complex (PDH):

It is an irreversible process !!
- PDH complex essentially converts pyruvate (3C) to acetyl Co-A (2C), yielding CO2 and NADH; it has: 3 Enzymes 5 Cofactors: Thiamine Pyrophosphate (TPP), Lipoic Acid, NAD+, FAD, Coenzyme A (CoA-SH)
(note that four different vitamins are involved: thiamine (B1), riboflavin (B2), niacin (B3), and pantothenate (B5))
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Coenzyme A and acetylCoA

- structure: 3-phosphoadenosinediphosphate + pantothenic acid + mercaptoethanolamine:

- acetylCoA is more energy-rich than ATP:

Lipoic acid: key cofactor

- Lipoic acid (acting attached on Lys sidechain of dihydrolipoyl transacetylase) can undergo redox reactions and can attach acetyl groups (similar to CoA):

Oxidation of pyruvate to acetylCoA

- five consecutive steps, achieved in PDH complex:

(also called pyruvate decarboxylase)

- PDH complex can be covalently modified by phosphorylation which makes it less active and dephosphorylation to make it more active. These processes are catalyzed by PDH-kinase and PDHphosphatase. An inherited decreased activity of PDH results in Chronic Lactic Acidosis (CLA). 7

Oxidation of pyruvate to acetylCoA

- PDH complex: supramolecular enzymatic complex of nanometric dimensions containing: E1 pyruvate dehydrogenase (aka pyruvate decarboxylase) E2 dihydrolipoyl transacetylase (20 trimers = 60 molecules) E3 dihydrolipoyl dehydrogenase - the swinging lipoyl arm of E2 (blue) can reach the active sites of E1 and E3 (enzymes are mechanically coupled!!!):

Decarboxylation of pyruvate
- achieved using TPP (vit B1) as cofactor, which carries the product = acetaldehyde:
Thiamine NH2 N H3C N H3C N S H pyrophosphate O O O P O P O O O

E1 =decarboxylase

E1

E2 =transacetylase

TPP
H3C R N S E1 H3C R O H3C C C O O N

Electron "Sink"
CO2 E1 S O O H3C H3C R N C S E1 Hydroxyethyl TPP H3C R N S E1

H3C C C OH

OH Enz-Base O

H3C C OH H S S
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Pyruvate
(see also the fate of pyruvate in glycolysis)

E2

Lys

HN C (CH2)4

Oxidation of activated acetaldehyde to acetate

- the active acetaldehyde bound on TPP is then transferred to the lipoyl moiety of dihydrolipoyl transacetylase (E2), where it undergoes an internal redox reaction: (contd)

Free thiamine released to accept another pyruvate

SCoA H3C C O O E2LysHN C (CH2)4 S SH O E2LysHN C (CH2)4

H3C HS .. CoA H3C C O S SH O E2 Lys HN C (CH2)4 R N S

E1

H3C C OH S SH

Acetyl-S-CoA
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2C

4C

6C

4C Citric acid cycle

6C

4C

6C

4C 4C 5C
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Citric acid cycle

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Citric acid cycle

Analogous to PDH Complex

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Citric acid cycle

Membrane Bound

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Citric acid cycle

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4-6 6 6 6 4 6

36-38 We will use 3 ATP for each NADH and 2 ATP for each FADH2
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TCA is amphibolic: anabolic and catabolic

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(anaplerotic = to fill up (gr.)

(concentration of various intermediates in citric acid cycle is kept ~ constant through anaplerotic reactions)
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Anaplerotic reaction: Pyruvate Carboxylase and use of Biotin as a 1 carbon carrier.

Note: the protein avidin (found in raw egg whites) strongly binds to biotin and can cause biotin deficiency. Dont eat too many raw eggs !!!
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Biological Tethers
- carry 1 or 2 carbon intermediates from one active site to another:

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Regulation of TCA and metabolic flow

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Fate of Citrate

Citrate Shuttle

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Sources and Fate of Succinate

Heme

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Goals and Objectives

Upon completion of this series of lectures at minimum you should be able to answer the following: What is cellular respiration, what are its stages, what are the main intermediates and enzymes involved? What is the ultimate electron acceptor in vivo, and which compounds are used in vivo for energy storage? Which are the main structural particularities of mitochondria and where are various enzymes relevant for cellular respiration placed? Which are the main steps, intermediates, and enzyme/enzyme complexes involved in oxidation of pyruvate to acetyl-CoA, what are their characteristics? What are the main steps, intermediates, and enzymes involved in the citric acid cycle, what are their characteristics, how is it regulated, and by which chemical entities, what decides the fate of citrate, pyruvate, succinate ? At which steps in the conversion of a molecule of glucose through glycolysis, coupled with cellular respiration do we consume/produce energy and what is the overall balance? Why do we state that the citric acid cycle is amphibolic, what are anaplerotic reactions (with examples), what is their significance? How is the citric acid coupled with other important physiologic processes?
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