The Pyruvate Dehydrogenase complex catalyzes the conversion of pyruvate to acetyl-CoA and includes three enzymes: E1, E2, and E3. E1 uses thiamine pyrophosphate to oxidize pyruvate and transfer the acetyl group to E2. Lipoic acid covalently attached to E2 carries the acetyl group to E3, where it is transferred to CoA to form acetyl-CoA. Electrons are transferred through the complex to NAD+ to form NADH. The complex is regulated by phosphorylation/dephosphorylation of E1 and by product inhibition by NADH and acetyl-CoA. Acetyl-CoA produced

1. Pyruvate Dehydrogenase
Copyright © 1999-2006 by Joyce J. Diwan.
All rights reserved.
Molecular Biochemistry I

2.  The matrix contains Pyruvate Dehydrogenase,
enzymes of Krebs Cycle, and other pathways, e.g.,
fatty acid oxidation & amino acid metabolism.
 The outer membrane contains large VDAC channels,
similar to bacterial porin channels, making the outer
membrane leaky to ions & small molecules.
matrix
inner
membrane
outer
membrane
inter-
membrane
space
mitochondrion
cristae
Glycolysis occurs in
the cytosol of cells.
Pyruvate enters the
mitochondrion to be
metabolized further.
Mitochondrial
Compartments:

3. It contains various transport catalysts, including a carrier
protein that allows pyruvate to enter the matrix.
It is highly convoluted, with infoldings called cristae.
Embedded in the inner membrane are constituents of the
respiratory chain and ATP Synthase.
matrix
inner
membrane
outer
membrane
inter-
membrane
space
mitochondrion
cristae
The inner
membrane is the
major permeability
barrier of the
mitochondrion.

4. Pyruvate Dehydrogenase, catalyzes oxidative
decarboxylation of pyruvate, to form acetyl-CoA.
H3C C C O−
O O
C S
O
H3C CoA
HSCoA
NAD+
NADH
+ CO2
Pyruvate Dehydrogenase
pyruvate acetyl-CoA

5.  The inner core of mammalian Pyruvate Dehydrogenase
is an icosahedral structure consisting of 60 copies of E2
.
 At the periphery of the complex are:
• 30 copies of E1
(itself a tetramer with subunits α2β2).
• 12 copies of E3
(a homodimer), plus 12 copies of an
E3 binding protein that links E3 to E2.
Pyruvate
Dehydrogenase: a large
complex containing many
copies of each of 3
enzymes, E1, E2, & E3.

6. Pyruvate Dehydrogenase Subunits
Enzyme Abbreviated Prosthetic Group
Pyruvate
Dehydrogenase
E1
Thiamine
pyrophosphate (TPP)
Dihydrolipoyl
Transacetylase
E2
Lipoamide
Dihydrolipoyl
Dehydrogenase
E3
FAD

7. FAD (Flavin Adenine Dinucleotide is derived from the
vitamin riboflavin. The dimethylisoalloxazine ring system
undergoes oxidation/reduction.
FAD is a prosthetic group, permanently part of E3.
Reaction: FAD + 2 e-
+ 2 H+
 FADH2
C
C
C
H
C
C
H
C
N
C
C
N
N
C
NH
C
H3C
H3C
O
O
CH2
HC
HC
HC
H2C
OH
O P O P O
O
O-
O
O-
Ribose
OH
OH
Adenine
C
C
C
H
C
C
H
C
N
C
C
H
N
N
H
C
NH
C
H3C
H3C
O
O
CH2
HC
HC
HC
H2C
OH
O P O P O
O
O-
O
O-
Ribose
OH
OH
Adenine
FAD FADH2
2 e−
+ 2 H+
dimethylisoalloxazine

8. Thiamine pyrophosphate (TPP) is a derivative of
thiamine (vitamin B1).
Nutritional deficiency of thiamine leads to the disease
beriberi.
Beriberi affects especially the brain, because TPP is
required for CHO metabolism, & the brain depends on
glucose metabolism for energy.
thiamine pyrophosphate (TPP)
N
NH3C NH2
CH2
S
C
N
H3C
CH2 O P O P O−
O−
O−
CH2
H
O O
+
acidic H+

9. Thiamine pyrophosphate (TPP) mechanism:
H+
readily dissociates from the C between N
and S in the thiazole ring.
The resulting carbanion (ylid) can attack the
electron-deficient keto carbon of pyruvate.
thiamine pyrophosphate (TPP)
N
NH3C NH2
CH2
S
C
N
H3C
CH2 O P O P O−
O−
O−
CH2
H
O O
+
acidic H+
C
C
CH3
O−
O
O
pyruvate

10. Lipoamide
includes a
dithiol that
undergoes
oxidation/
reduction.
S CH2
CH2
CHS
CH2 CH2 CH2 CH2 C NH (CH2)4 CH
NH
C O
O
HS CH2
CH2
CHHS
CH2 CH2 CH2 CH2 C NH (CH2)4 CH
NH
C O
O
2e−
+ 2H+
lipoamide
dihydrolipoamide
lysinelipoic acid

11. The carboxyl at the end of lipoic acid's hydrocarbon chain
forms an amide bond to the side-chain amino group of a
lysine residue of E2
, yielding lipoamide.
A long flexible arm, including hydrocarbon chains of
lipoate and the lysine R-group, links each lipoamide dithiol
group to one of 2 lipoate-binding domains of each E2.
Lipoate-binding domains are themselves part of a flexible
strand of E2 that extends out from the core of the complex.
S CH2
CH2
CHS
CH2 CH2 CH2 CH2 C NH (CH2)4 CH
NH
C O
O
HS CH2
CH2
CHHS
CH2 CH2 CH2 CH2 C NH (CH2)4 CH
NH
C O
O
2e−
+ 2H+
lipoamide
lysinelipoic acid

12. The long flexible attachment allows lipoamide functional
groups to swing between E2 active sites in the core of the
complex & active sites of E1 & E3 in the outer shell.
E3 binding protein that binds E3 to E2 also has attached
lipoamide that can exchange of reducing equivalents with
lipoamide on E2.
Diagrams in: website of the laboratory of Wim Hol
article by Milne et al. (Fig. 5)
S CH2
CH2
CHS
CH2 CH2 CH2 CH2 C NH (CH2)4 CH
NH
C O
O
HS CH2
CH2
CHHS
CH2 CH2 CH2 CH2 C NH (CH2)4 CH
NH
C O
O
2e−
+ 2H+
lipoamide
lysinelipoic acid

13. Organic arsenicals are potent inhibitors of lipoamide-
containing enzymes such as Pyruvate Dehydrogenase.
These highly toxic compounds react with “vicinal”
dithiols such as the functional group of lipoamide.
HS
HS
R
S
S
R
R' As O AsR'+
H2O

14. The final electron acceptor
is NAD+
.
Coenzyme A-SH + HO C
O
CH3
Coenzyme A-S C
O
CH3 + H2O
acetic acid
acetyl-CoA
N
R
H
C
NH2
O
N
R
C
NH2
O
H H
+
2e−
+ H
+
NAD+
NADH
In the overall reaction
catalyzed by the Pyruvate
Dehydrogenase complex,
the acetic acid generated
is transferred to
coenzyme A.

15. Sequence of reactions catalyzed by Pyruvate
Dehydrogenase complex:
1. The keto C of pyruvate reacts with the carbanion of
TPP on E1
to yield an addition compound.
The electron-pulling (+) charged N of the thiazole ring
promotes CO2
loss. Hydroxyethyl-TPP remains.
2. The hydroxyethyl carbanion on TPP of E1
reacts with
the disulfide of lipoamide on E2
. What was the keto C
of pyruvate is oxidized to a carboxylic acid, as the
lipoamide disulfide is reduced to a dithiol.
The acetate formed by oxidation of the hydroxyethyl
is linked to one of the thiols of the reduced lipoamide
as a thioester (~).

16. Sequence of reactions (continued)
3. Acetate is transferred from the thiol of lipoamide
to the thiol of coenzyme A, yielding acetyl CoA.
4. The reduced lipoamide, swings over to the E3
active site.
Dihydrolipoamide is reoxidized to the disulfide, as
2 e−
+ 2 H+
are transferred to a disulfide on E3
(disulfide interchange).
3. The dithiol on E3
is reoxidized as 2 e-
+ 2 H+
are
transferred to FAD.
The resulting FADH2
is reoxidized by electron
transfer to NAD+
, to yield NADH + H+
.

17. Acetyl CoA, a product of the Pyruvate Dehydrogenase
reaction, is a central compound in metabolism.
The "high energy" thioester linkage makes it an
excellent donor of the acetate moiety.
acetyl-coenzyme A
H3C C
O
S CoA
View an animation of the Pyruvate Dehydrogenase
reaction sequence.

18. Acetyl CoA functions as:
 input to Krebs Cycle, where the acetate moiety is
further degraded to CO2.
 donor of acetate for synthesis of fatty acids, ketone
bodies, & cholesterol.
glucose-6-P
Glycolysis
pyruvate
fatty acids
acetyl CoA ketone bodies
cholesterol
oxaloacetate citrate
Krebs Cycle

19. Regulation of Pyruvate Dehydrogenase Complex:
Product inhibition by NADH & acetyl CoA:
 NADH competes with NAD+
for binding to E3
.
 Acetyl CoA competes with CoA for binding to E2
.

20. Regulation by E1 phosphorylation/dephosphorylation:
Specific regulatory Kinases & Phosphatases associated
with Pyruvate Dehydrogenase in the mitochondrial matrix:
 Pyruvate Dehydrogenase Kinases catalyze
phosphorylation of serine residues of E1, inhibiting
the complex.
 Pyruvate Dehydrogenase Phosphatases reverse this
inhibition.
Pyruvate Dehydrogenase Kinases are activated by NADH
& acetyl-CoA, providing another way the 2 major products
of Pyruvate Dehydrogenase reaction inhibit the complex.
Kinase activation involves interaction with E2 subunits to
sense changes in oxidation state & acetylation of lipoamide
caused by NADH & acetyl-CoA.

21. During starvation:
 Pyruvate Dehydrogenase Kinase increases in
amount in most tissues, including skeletal muscle, via
increased gene transcription.
 Under the same conditions, the amount of Pyruvate
Dehydrogenase Phosphatase decreases.
The resulting inhibition of Pyruvate Dehydrogenase
prevents muscle and other tissues from catabolizing
glucose & gluconeogenesis precursors.
 Metabolism shifts toward fat utilization.
 Muscle protein breakdown to supply gluconeogenesis
precursors is minimized.
 Available glucose is spared for use by the brain.

22. The increased cytosolic Ca++
that occurs during
activation of muscle contraction can lead to Ca++
uptake
by mitochondria.
The higher Ca++
stimulates the phosphatase, &
dephosphorylation activates Pyruvate Dehydrogenase.
Thus mitochondrial metabolism may be stimulated
during exercise.
Pyruvate
Dehydro-
genase
in matrix
mitochondrion Ca++
A Ca++
-sensitive isoform of
the phosphatase that
removes Pi
from E1
is
expressed in muscle.

23.  Lecture notes relating to Krebs Cycle are not
provided, because students will present the lectures.
However see the summary of Krebs Cycle
embedded in the class web page.
 Some questions on Krebs Cycle are included in the
self-study quiz for this class.