Download The Aerobic Fate of Pyruvate

The Aerobic Fate of Pyruvate
February 12, 2003
Bryant Miles
I could tell that some of you were not impressed by the mere 2 ATPs produced per glucose by glycolysis.
The 2 ATP’s produced are only a small fraction of the potential energy available from glucose. Under
anaerobic conditions, animals convert glucose into 2 molecules of lactate. Much of the potential energy
of the glucose molecule remains untapped. Under Aerobic conditions a much more dynamic pyruvate
metabolism occurs. The 2 moles of NADH produced by glyceraldehyde-3-phosphate dehydrogenase are
oxidized in the electron transport chain back to NAD+. The electron transport chain generates a proton
gradient that drives the synthesis of 5 ATP molecules from ADP and Pi. Further more, the pyruvate
formed by glycolysis is converted to acetyl-CoA by pyruvate dehydrogenase (generating another 2 moles
of NADH per glucose and another 5 ATPs by oxidative phosphorylation). The acetyl-CoA formed enters
into the citric acid cycle where it is completely oxidized into CO2. The electrons liberated by oxidation
are captured by NAD+ or FAD which are then transferred via the electron transport chain ultimately to O2,
the final electron acceptor. The electron transport chain is coupled to generating a proton gradient which
produces a proton motive force that drives
the synthesis of ATP. This allows the net
production of 32 molecules of ATP to be
formed per glucose molecule. The function
of the citric acid cycle is to harvest high
energy electrons from carbon fuels. The
citric acid cycle is the central metabolic hub
of the cell, the gateway of aerobic
metabolism. The citric acid cycle produces
intermediates which are precursors for fatty
acids, amino acids, nucleotide bases,
cholesterol and porphoryins.
The citric acid cycle is shown on the next page. The citric acid cycle occurs in the mitochondria of
eukaryotes. New carbons enter the citric acid cycle through acetyl-CoA. The acetyl group may come
from pyruvate, fatty acids, ketobodies, ethanol or alanine. The two carbons of acetyl-CoA are transferred
to oxaloacetate to yield the first tricarboxylic acid citrate in a reaction catalyzed by citrate synthase. A
dehydration followed by a rehydration rearranges citrate into isocitrate. Two successive decarboxylations
coupled to the generation of 2 NADH produce succinyl-CoA. Four steps later oxaloacetate is regenerated
along with a GTP, FADH2 and NADH.
O
C
Cα
Cβ
Cleavage site
The citric acid cycle may seem like an elaborate way to oxidize acetate into carbon
dioxide, but there is chemical logic to the cycle. In order to directly oxidize acetate
into two molecules of CO2 a C—C bond must be broken. Under the mild
conditions found in cells, there is insufficient energy to break the bond. Biological
systems often break C—C bonds between carbon atoms α and β to a carbonyl
group. Examples are aldolase and transaldolase.
O
OH
C
Cα
Cβ
Cleavage site
Another common type of C—C cleavage is α-cleavage of an α-hydroxy-ketone
which we have seen before in transketolase and pyruvate decarboxylase, two TPP
dependent enzymes.
Neither of these common strategies for cleavage of C—C bonds is available to
acetate. Instead the acetate is activated in the form acetyl-CoA, condensed with
oxaloacetate to form citrate and then carrying out a β-cleavage in subsequent steps.
The Net reaction of the citric acid cycle is:
3NAD+ + FAD + GDP + Pi + Acetyl-CoA + 2H2O
3NADH + FADH2 + GTP + CoA + 2CO2 + 3H+
The Aerobic Fate of Pyruvate
In eukaryotes, the reactions of the citric acid cycle occur in
the mitochondria. The mitochondrian is enclosed by a
double membrane. All of the glycolyitic enzymes are
found in the cytosol of the cell. Charged molecules such as
pyruvate must be transported in and out of the
mitochondria. Small charged molecules with molecular
weights of less than 10,000 can freely diffuse through the
outer mitochondrial membrane through aqueous channels called porins. A transport protein called
pyruvate translocase specifically transports pyruvate through the inner mitochondrial membrane into the
matrix of the mitochondrian.
Once in the mitochondria, pyruvate dehydrogenase converts pyruvate into acetyl-CoA and CO2.
-
O
O
O
C
C
NAD=
CH3
NADH + CO2
O
+
S
S
CH2
Pyruvate Dehydrogenase
NH
CH2
C
NH
O
CH2
O
CH2
CH2
NH
CH2
NH
O
HO
C
H
H3C
C
CH3
O
N
O
P
O-
N
O
O
P
N
N
NH2
C
O
HO
C
H
H3C
C
CH3
NH 2
C
H2C
CH3
CH2
H
CH2
C
C
H2C
O
N
O
P
O-
O
N
O
O
P
O
H
O
OH
H
O
H
P
H
H
O
OH
H
H
O-
N
O
O-
O
O-
N
O
H
P
O-
O-
O-
Coenzyme A
Acetyl Coenzyme A
This reaction may look straight forward but it is complex. Pyruvate dehydrogenase is a humongous
enzyme that can be visualized by electron micrographs. See Figure 16-3 of text book. Pyruvate
dehydrogenase is a multienzyme complex held together by noncovalent interactions. There are three
different enzymes and five coenzymes. The three individual enzymes are E1, pyruvate dehydrogenase, E2,
dihydrolipoamide transacetylase, and E3, dihyrolipoamide dehydrogenase. The multienzyme complex
consists of 24 subunits of E1, 24 subunits of E2 and 12 subunits of E3. In E1 we find a thiamine
pyrophosphate coenzyme. In E2 the is a lipoic acid coenzyme. In E3 there is a FAD coenzyme. The
molecular weight of pyruvate dehydrogenase isolated from E. coli is 4.6 million Daltons, slightly larger
than a ribosome. In mammals, pyruvate dehydrogenase is twice that size and has two additional
regulatory subunits. One is a protein kinase which phosphorylates three serine residues in E1, the other is
a phosphatase which hydrolytically removes the same phosphoryl groups from the serines of E1.
The structure of the pyruvate dehydrogenase complex is shown above. The E2, dihydrolipoamide
transacetylase are shown as red balls. These 24 subunits create the core upon which the pyruvate
dehydrogenase complex is built. The E1, pyruvate dehydrogenase enzyme is an α2β2 dimer shown as the
orange and yellow balls. There are 24 of the αβ dimers in the complex. There are 12 subunits of the E3,
dihyrolipoamide dehydrogenase complex shown in purple. The E3 subunits associate into dimers, so
there are six E3 dimers in the complex.
Lets look at the chemistry going on in this complex.
R'
R'
+
R
In E1, pyruvate dehydrogenase enzyme, we have a tightly
bound thiamine pyrophosphate coenzyme or prosthetic group.
We have encountered this cofactor before, pyruvate
decarboxylase and transketolase. The reaction catalyzed in
this subunit is similar to that of pyruvate decarboxylase.
H3 C
H3 C
+
R
N
N
S
C
S
:
-C
H
-
O
C
C
O
O
CH3
The next step in the enzyme catalyzed reaction involves a
new cofactor we haven’t encountered yet. It is lipoic acid.
H
B
H3C
H3 C
R'
R'
R
+
R
C
S
C
O
C
C
O
O
S
S
H
H
S
C
-
S
N
CO2
N
S
CH3
CH3
O
O
O
-
O
H
H
HN
Lipoic Acid
:B
H3C
H3C
R'
+
R
N
R
C
H
C
R
'
+
S
CH3
C
B
H
H
N
-
:C
O
O
H
H
S
N
H
O
CH3
Lipoamide
Lipoic acid is not found free in nature. Instead lipoic acid is covalently attached to a lysine residue
through an amide linkage. The enzyme that catalyzes the formation of the lipoamide linkage uses ATP to
generate AMP and pyrophosphate. Lipoic acid is an important cofactor because it couples acyl-group
transfers with electron transfers during the oxidation and decarboxylation of α-keto acids. Because the
ring strain inherent in the disulfide cyclic structure is relieved upon reduction, lipoic acid has a strong
negative reduction potential, Eo’ = −0.30 V.
2e- + 2H+
R
S
R
H
S
H
SH HS
Eo' = -0.30 V
H3C
H3C
R'
+
R
N
R
C
H
N
S
C
B:
R'
S
C
CH3
C
O
O
H
H
CH3
H3C
H3C
R'
R'
R
R
N
C
S
C
SH
O
S
C
CH3
-
S
H
R
B:
B
H3C
R'
R
C
SH
O
CH3
+
R
N
- C
:
S
S
:C
O
S
H
R
N
H
S
CH3
To summarize the chemistry up to this point, This enzyme has taken pyruvate, used TPP to catalyze the
decarboxylation of an α-ketoacid to form the hydroxyethyl-TPP intermediate which is stable enough to
isolate. The hydroxyethyl-TPP was then oxidized by and concomitantly transferred to lipoamide to form
an acetyllipoamide. The acetyllipoamide is the final product of E1. The lipoamide prosthetic group is
part of the E2 Dihydrolipoyl transacetylase enzyme. The next step in the course of the reaction
mechanism occurs in the active site of the E2, Dihydrolipoyl transacetylase enzyme, which catalyzes the
transfer of the acetyl group from lipoamide to coenzyme A.
H
S
CoA
O
S
C
SH
O
SH
CH3
SH
+
H3C
C
S
CoA
R
R
If you look back to the structure of lipoamide, you will see it has a long flexible tether linking the amide
to the E2 enzyme. The length of this tether is about 45 Å. This is long enough to reach the active site of
E1 to form the acetyllipoamide and then carry this intermediate to the active site of the E2 enzyme to form
acetyl-CoA and the dihydrolipoamide which is then carried to yet the third active site, the E3,
dihyrolipoamide dehydrogenase enzyme.
R
NH2
N
N
FMN
O
H2C
P
O
H
C
OH
H
C
OH
H
C
OH
N
O
O
P
O
O
O-
O-
H
H
OH
OH
H
N
FAD
Quinone
H3C
N
H3C
N
N
H
O
H3C
N
H
N
O
NH
CH2
H3C
N
.
O
NH
R
O
FAD
H3C
N
H3C
N
FADH
Semiquinone
The E3, dihyrolipoamide dehydrogenase enzyme regenerates lipoamide for
another round of catalysis. This enzyme is a flavoprotein, meaning, it has a
tightly bound FAD prosthetic group. Flavins undergo 2 electron reductions,
but can accept electrons one at a time,unlike NAD+ which can only accept
two electrons at a time in the form of a hydride. This gives flavoproteins
more catalytic diversity than NAD coenzymes.
FADH2
Hydroquinone
N
.
H
H3C
N
H3C
N
NH
O
H
R
O
.
H
N
O
NH
H
O
Dihydrolipoyl dehydrogenase
SH
SH
R
S
+
FAD
+
FADH2
S
R
The final step of the reaction catalyzed by this E3 enzyme is the transfer of the 2 electrons from the tightly
bound FADH2 to the transiently bound NAD+ to generate NADH + H+.
FADH2 + NAD+ FAD + NADH + H+
This is a usual electron transfer. The common role of FAD is to accept electrons from NADH. One thing
about flavoproteins is that the protein bound flavins have a great variety of reduction potentials. In this
enzyme the reduction potential is shifted such that FADH2 is the electron donor and NAD+ is the acceptor.
Typical flavoprotein
FAD + 2e- + 2H+ FADH2 Eo’≈ 0 V
NAD + 2e- + H+ NADH
Eo’ = -0.315 V