Download Pyruvate Dehydrogenase

Molecular Biochemistry I
Pyruvate Dehydrogenase
Copyright © 1999-2006 by Joyce J. Diwan.
All rights reserved.
Glycolysis occurs in
the cytosol of cells.
Pyruvate enters the
mitochondrion to be
metabolized further.
Mitochondrial
Compartments:
matrix
cristae
intermembrane
space
inner
membrane mitochondrion
outer
membrane
 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
The inner
membrane is the
major permeability
barrier of the
mitochondrion.
cristae
intermembrane
space
inner
membrane mitochondrion
outer
membrane
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.
Pyruvate Dehydrogenase
H3C
O
O
C
C
pyruvate
HSCoA
O
O
H3C
NAD+ NADH
C
S
CoA
+ CO2
acetyl-CoA
Pyruvate Dehydrogenase, catalyzes oxidative
decarboxylation of pyruvate, to form acetyl-CoA.
Pyruvate
Dehydrogenase: a large
complex containing many
copies of each of 3
enzymes, E1, E2, & E3.
 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 a2b2).
• 12 copies of E3 (a homodimer), plus 12 copies of an
E3 binding protein that links E3 to E2.
Pyruvate Dehydrogenase Subunits
Enzyme
Abbreviated
Prosthetic Group
Pyruvate
Dehydrogenase
E1
Thiamine
pyrophosphate (TPP)
Dihydrolipoyl
Transacetylase
E2
Lipoamide
Dihydrolipoyl
Dehydrogenase
E3
FAD
dimethylisoalloxazine
O
H
C
C
N
O

H3C
C
C
C
NH
H3C
C
C
C
C
C
H
N
H
C
+
2e +2H
O
N
H
N
H3C
C
C
C
NH
H3C
C
C
C
C
C
H
CH2
FAD
N
O
N
H
CH2
HC
OH
HC
OH
HC
OH O
H2C
C
O
P
O-
Adenine
O
O
P
O
Ribose
FADH2
HC
OH
HC
OH
HC
OH O
H2C
O
O-
P
Adenine
O
O
O-
P
O
Ribose
O-
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
O
O
H3C
CH2
N
H3C
CH2
H
O
P
O
O
+
N
C
N
CH2
P
O
S
acidic H+
NH2
thiamine pyrophosphate (TPP)
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.
O
O
O
H3C
CH2
N
H3C
CH2
H
O
P
O
O
+
N
C
N
CH2
O
P
O
S
acidic H+
NH2
thiamine pyrophosphate (TPP)
Thiamine pyrophosphate (TPP) mechanism:
O
O
H+ readily dissociates from the C between N
and S in the thiazole ring.
C
The resulting carbanion (ylid) can attack the
electron-deficient keto carbon of pyruvate.
CH3
C
O
pyruvate
S
CH2
CH2
S
lipoic acid
CH
O
CH2 CH2 CH2 CH2 C
Lipoamide
includes a
dithiol that
undergoes
oxidation/
reduction.
lipoamide
lysine
NH
NH (CH2)4 CH
C
O
2e + 2H+
HS
CH2
CH2
HS
O
CH
CH2 CH2 CH2 CH2 C
dihydrolipoamide
NH
NH (CH2)4 CH
C
O
S
CH2
CH2
S
CH
lipoic acid
O
CH2 CH2 CH2 CH2 C
lipoamide
lysine
NH
NH (CH2)4 CH
C
O
2e + 2H+
The carboxyl at the end of lipoic acid's hydrocarbon chain
CH2
forms an HS
amide
bond to the side-chain amino group of a
lysine residue ofCHE2 2, yielding lipoamide.
NH
HS
CH
O
A long flexible arm,
including
hydrocarbon chains of
CH2 CH
2 CH2 CH2 C NH (CH2)4 CH
lipoate and the lysine R-group, links each lipoamide dithiol
C O
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
S
CH
lipoic acid
O
CH2 CH2 CH2 CH2 C
lipoamide
lysine
NH
NH (CH2)4 CH
C
O
2e + 2H+
The long flexible attachment allows lipoamide functional
CH2 between E2 active sites in the core of the
groups toHS
swing
CHsites
2
complex & active
of E1 & E3 in the outer
shell.
NH
HS
CH
O
E3 binding protein
that binds E3 to E2 also has attached
CH2 CH2 CH2 CH2 C NH (CH2)4 CH
lipoamide that can exchange of reducing equivalents with
C O
lipoamide on E2.
Diagrams in: website of the laboratory of Wim Hol
article by Milne et al. (Fig. 5)
H2O
HS
R'
As
O
S
R'
+
HS
As
S
R
R
Organic arsenicals are potent inhibitors of lipoamidecontaining enzymes such as Pyruvate Dehydrogenase.
These highly toxic compounds react with “vicinal”
dithiols such as the functional group of lipoamide.
O
C CH3
Coenzyme A-SH + HO
acetic acid
O
Coenzyme A-S
C
CH3 + H2O
acetyl-CoA
In the overall reaction
catalyzed by the Pyruvate
Dehydrogenase complex,
the acetic acid generated
is transferred to
coenzyme A.
H
O
H
H
C
The final electron acceptor
is NAD+.
C
NH2
+
N
O

NH2
+
2e + H
N
R
R
NAD+
NADH
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 (~).
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+.
View an animation of the Pyruvate Dehydrogenase
reaction sequence.
O
H3C
C
S
CoA
acetyl-coenzyme A
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.
glucose-6-P
Glycolysis
pyruvate
fatty acids
acetyl CoA
oxaloacetate
ketone bodies
cholesterol
citrate
Krebs Cycle
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.
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.
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.
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.
A Ca++-sensitive isoform
of the phosphatase that
removes Pi from E1 is
expressed in muscle.
Pyruvate
Dehydrogenase
in matrix
mitochondrion
Ca++
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.
 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.