CITRIC ACID CYCLE

-Pyruvate Dehydrogenase
Part I

Refer to: Lehninger Principles of Biochemistry (Chapter 16)

Dr Fawaz Awad; 2018

1
 OBJECTIVES
1. To understand how the pyruvate dehydrogenase complex
acts as a mediator of flow of carbon compounds between the
glycolytic pathway and the citric acid cycle.

2. To understand the central role of the citric acid cycle in

mitochondrial energy metabolism.

3. The structures of the intermediates in the cycle.

4. The names of the enzymes that catalyze each step.

5. The cofactors and products involved in each step.

2
 Pyruvate transport into mitochondria

• Pyruvate (has negative charge) generated in the

cytoplasm by glycolysis must be transported
across the inner mitochondrial membrane via a
pyruvate/H+ symport.
• This transport uses some of the energy (Active
transport) stored in the mitochondrial inner
membrane electrical potential gradient.

3
4
 Pyruvate dehydrogenase (PDH)
complex
Function of pyruvate dehydrogenase complex: Connection between cytoplasmic
glycolysis and mitochondrial citric acid cycle, which works only in the presence of
O2.

5
Catabolism of proteins, fats, and
carbohydrates in the three stages of
cellular respiration:

Stage 1: oxidation of fatty acids, glucose,

and some amino acids yields acetyl-CoA.

Stage 2: oxidation of acetyl groups in the

citric acid cycle includes four steps in
which electrons are abstracted.

Stage 3: electrons carried by NADH and

FADH2 are funneled into a chain of
mitochondrial electron carriers—the
respiratory chain—ultimately reducing O2
to H2O. This electron flow drives the
production of ATP.

6
Sources of acetyl-CoA in mitochondria

7
 Pyruvate Dehydrogenase (PDH)
Within the mitochondria, pyruvate is oxidative decarboxylated to acetyl-CoA.
This reaction is catalyzed by several different enzymes working sequentially in a multi-enzyme
complex collectively designated as the pyruvate dehydrogenase complex.

Overall reaction:

Pyruvate Dihydrolipoyl
Dihydrolipoyl
dehydrogenase dehdrgenase
transacetylase

• irreversible in mitochondria

Or Simply:
Pyruvate + NAD+ + CoA ® Acetyl-CoA + NADH + H+ + CO2 8
 Pyruvate Dehydrogenase Complex
• Contains three enzymes each present in multiple copies:
1. Pyruvate dehydrogenase (E1) (TPP): oxidative decarboxylation of pyruvate.
2. Dihydrolipoyl transacetylase (E2) (Lipoamide): transfer of the acyl group to
CoA.
3. Dihydrolipoyl dehydrogenase (E3) (FAD) regeneration of the oxidized
forms of lipoamide and transfer of electrons to NAD.

• In addition, five different coenzymes or prosthetic groups are needed:

1. Thiamine pyrophosphate (TPP=B1)
2. Flavin adenine dinucleotide (FAD= B2)
3. Coenzyme A (CoA). vitamin B5 derived.
4. Nicotinamide adenine dinucleotide (NAD=B3)
5. Lipoate

• All are clustered for efficient handling of intermediates 9

 Advantages of the multienzyme
complex
• The PDH complex is a classic multi enzyme complex in which a
series of chemical intermediates remain bound to the enzyme
molecules as a substrate is transformed into the final product.
• Five cofactors, four derived from vitamins, participate in the
reaction mechanism. The regulation of this enzyme complex also
illustrates how a combination of covalent modification and
allosteric regulation results in precisely regulated flux through a
metabolic step
• Higher rate of reaction: Because product of one enzyme acts as a
substrate of other, and is available for the active site of next
enzyme without much diffusion.
• Minimum side reaction.
• Coordinated control. 10
 Coenzyme A (CoASH)

vitamin B5

• Coenzyme A (CoA). A hydroxyl group of pantothenic acid is joined to a modified ADP

moiety by a phosphate ester bond, and its carboxyl group is attached to b-
mercaptoethylamine in amide linkage.
• The hydroxyl group at the 3´position of the ADP moiety has a phosphoryl group not
present in ADP itself.
• The thiol -SH group of the mercaptoethylamine moiety forms a thioester with acetate in
acetyl-coenzyme A (acetyl-CoA).
• Act as acceptor and donor of the acyl group. not free transported across cellular
membrane. 11
 Lipoic acid (lipoate)
• Lipoic acid (lipoate) in amide linkage with
the side chain (ε-amino group) of a Lys
residue.

• The lipoyllysyl moiety is the prosthetic

group of dihydrolipoyl transacetylase (E2 of
the pyruvate dehydrogenase complex).

• The lipoyl group (has two thiol) occurs in

oxidized (disulfide) and reduced (dithiol)
forms and acts as a carrier of both
hydrogen and an acetyl (or other acyl)
group.

• The attachment of lipoate to the end of a

Lys side chain in E2 produces a long,
flexible arm that can move from the active
site of E1 to the active sites of E2 and E3
12
2

13
Oxidative decarboxylation of pyruvate to acetyl-CoA
by the PDH complex
First step

The first step is the slowest and therefore limits the rate of the overall reaction. It is also 14
the
point at which the PDH complex exercises its substrate specificity
 Substrate channeling
Ø Pyruvate reacts with thiamine pyrophosphate bound to E1. Pyruvate
undergoes decarboxylation to the hydroxyl ethyl derivative, with a loss of
CO2.

Ø The acetyl group and 2 electrons from TPP are transferred to the oxidized
form of the lipoyllysyl group of the core enzyme (E2).

Ø A transesterification occurs where the -SH group of CoA replaces the -SH
group of E2 to yield acetyl-CoA and the reduced form of the lipoyl group.

Ø E3 transfers two hydrogen from the reduced lipoyl groups of E2 to FAD.

Ø The reduced FADH2 of E3 transfers a hydride ion to NAD+, forming NADH.

15
 Beriberi disease
Pyruvate decarboxylase depends
on cofactors thiamine pyrophosphate (TPP) and
magnesium.

Beriberi is a disease caused by a lack of vitamin

B1 (thiamine) in the body.

Accumulate of body fluids , pain, paralysis

…death

17