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PDH
17.1
What Are the Metabolic Fates of NADH and
Pyruvate Produced in Glycolysis?
NADH is energy - two possible fates:
If O2 is available:
NADH is re-oxidized in the electron transport pathway, making
ATP in oxidative phosphorylation
If O2 is not available (anaerobic conditions):
NADH is re-oxidized by lactate dehydrogenase (LDH),
providing additional NAD+ for more glycolysis
What Are the Metabolic Fates of NADH and
Pyruvate Produced in Glycolysis?
Pyruvate is also energy - two possible fates:
If O2 is available
pyruvate enters the mitochondria, where it undergoes further
breakdown
If O2 is not available (anaerobic conditions) fermentation
occurs and pyruvate undergoes reduction
Fermentation is an anaeorbic process and does not require
oxygen.
In humans, pyruvate is reduced to lactic acid during
fermentation.
To enter the Krebs cycle, pyruvic acid from glycolysis
must first be “prepped” into a usable form, Acetyl-CoA
CoA
2
Acetic
acid
1
Pyruvic
acid
CO2
3
Acetyl-CoA
(acetyl-coenzyme A)
Coenzyme A
Figure 6.10
Transition Reaction
Pyruvic Acid  AcetylCoA + CO2 + NADH
Transition reaction – pyruvate is oxidized to a 2-carbon acetyl group
carried by CoA, and CO2 is removed
- occurs twice per glucose molecule
Mitochondria: powerhouse
A mitochondrion is a cellular organelle that has a
double membrane, with an intermembrane space
between the two layers.
The transition reaction and citric acid cycle occur in
the mitochondrial matrix.
The electron transport system is located in the cristae
of the mitochondria.
The formation of acetyl
CoA from pyruvate is a
key irreversible step in
animals
Animals are unable to convert
acetyl CoA into glucose
The oxidative decarboxylation
of pyruvate into acetyl-CoA
commits the carbon atoms of
glucose to two principle fates
-oxidation to CO2 by the citric
acid cycle (and energy
generation)
-incorporation into lipid
Entry of Pyruvate into the Mitochondrion
Pyruvate translocase transports pyruvate into the mitochondria in
symport with H+
Symport: is used to denote an integral membrane protein that
simultaneouly transports two substances across membrane in the
same direction.
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 complex consists of three distinct enzymes
(in addition to the five coenzymes)
This reaction involves
--generation of a reduced electron carrier (NADH)
--decarboxylation of pyruvate
--metabolic activation of the remaining two carbons of pyruvate
the three enzymes involved are assembled into a highly organized
multienzyme assembly called the pyruvate dehydrogenase complex
Conversion of Pyruvate to Acetyl CoA
Pyruvate dehydrogenase complex (PDH complex) is a
multienzyme complex containing:
3 enzymes + 5 coenzymes + other proteins
(+ ATP coenzyme as a regulator)
E1 = pyruvate dehydrogenase
E2 = dihydrolipoamide acetyltransferase
E3 = dihydrolipoamide dehydrogenase
Structure of the PDH complex
(a) Core of the complex
(24 E chains)
(b) Model of the entire complex: 12 E1
dimers (blue), 12 E2 dimers (yellow),
6 E3 dimers (green) surround the
core
Pyruvate dehydrogenase complex in E. coli
Pyruvate Dehydrogenase Subunits
Enzyme
Abbreviated
Prosthetic Group
Pyruvate
Dehydrogenase
E1
Thiamine
pyrophosphate (TPP)
Dihydrolipoyl
Transacetylase
E2
Lipoamide
Dihydrolipoyl
Dehydrogenase
E3
FAD
Pyruvate dehydrogenase complex - mechanism
1. E1 accepts a two-carbon
aldehyde group from pyruvate and
binds it to TPP forming
hydroxyethyl TPP
2. Aldehyde group on TPP
is transferred by E1 to the
first lipoamide swinging
arm on E2 and
simultaneously oxidized
to an acetyl group
6. Reduced FADH2 is
oxidized by NAD+
5. E3 oxidizes the reduced
lipoamide by transferring
two hydrogens to FAD
3. Acetyl group is tranferred
to the second swinging arm
of lipoamide which
positions it for transfer to
CoA
4. Acetyl group is transferred to CoA producing acetyl-CoA
http://www.uwsp.edu/chemistry/tzamis/ch260/pyrdh2aseanim.gif
O
OH
OH
CH3 -C
CH3 -C -COOHOH CH3 -C
CoASH
CoASH
CH3 -C TDP
Pyruvate
CO2
O
CH3 -C-S-CoASH
Acetyl CoA
FAD
CO2
CO2
E1
E2
E3
CO2
Lipoate
NAD+
HS
S
TDP = thiamine diphosphate
SH
S
substrates
products
prosthetic group
coenzymes
O
CH3 -C-S-CoA
Acetyl CoA
TDP
E1
E3
E2
CO2
FADH2
Lipoate
S
S
NAD+
TDP = thiamine diphosphate
substrates
products
prosthetic group
coenzymes
O
CH3 -C-S-CoA
Acetyl CoA
TDP
FAD
E1
E2
E3
NADH
CO2
Lipoate
S
S
NAD+
TDP = thiamine diphosphate
substrates
products
prosthetic group
coenzymes
O
O
CH3 -C-S-CoA
CH3 -C -COOH
Pyruvate
CoASH
TDP
Acetyl CoA
FAD
E1
E2
E3
NADH
CO2
Lipoate
S
S
NAD+
Inhibition of lipoamide by arsenic and mercury
Arsenic poisoning
Arsenite complexes with
lipoamide – inactivating it
Some sulfhydral reagents relieve the
inhibition by forming a complex with
the arsenite that can be excreted
(competitive inhibition)
Mercury poisons in the same way
Thiamine deficiency (B1)
What would be the results of a diet deficient in thiamine?
Thiamine pyrophosphate is a prosthetic group of three important enzymes
• pyruvate dehydrogenase
• a-ketoglutarate dehydrogenase (CAC)
• transketolase (pentose phosphate pathway)
If the pyruvate dehydrogenase complex were unable to catalyze the formation
of acetyl-CoA from pyruvate – what would accumulate?
In cells that rely on glucose for fuel (do not use fats) – the energy that is
provided when pyruvate is converted to acetyl-CoA is not generated
Which cells in the body rely primarily on glucose for energy?
Cells of the nervous system and heart, therefore neurologic and cardiac
symptoms are associated with a thiamine deficiency
Formation of Acetyl Coenzyme A from pyruvate: Summary
this occurs in the mitochondrial matrix
and is the link between glycolysis
and the citric acid cycle
under aerobic conditions, acetyl coenzyme A is formed in the mitochondria by the oxidative
decarboxylation of pyruvate
pyruvate + NAD+ + CoA
acetyl CoA + CO2 + NADH
this reaction is catalyzed by pyruvate dehydrogenase
the NAD+ required for this reaction and for the oxidation of glyceraldehyde 3 phosphate is
regenerated when NADH ultimately transfers its electrons to O2 through the electron transport
chain in mitochondria
In the overall reaction, the carboxyl group of pyruvate is lost as CO2 while the remaining two
carbons form the acetyl moiety of acetyl-CoA
generation of a reduced electron carrier (NADH)
this reaction is highly exergonic and is essentially irreversible in vivo
∆G0’ = - 33.5 kJ/mol
S
CH2
CH2
S
lipoic acid
CH
O
CH2 CH2 CH2 CH2 C
lipoamide
Lipoamide
includes a
dithiol that
undergoes
oxidation/
reduction.
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
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-
O
Ribose
FADH2
HC
OH
HC
OH
HC
OH O
H2C
O
P
O-
Adenine
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
phosphorylation by pyruvate dehydrogenase kinase, (on E1) switches off the
activity of the complex
dephosphorylation by pyruvate dehydrogenase phosphatase restores activity of
the complex
Both the kinase and the phosphatase are bound to E2
Kinase activation involves interaction with E2 subunits to sense changes in
oxidation state & acetylation of lipoamide caused by NADH & acetyl-CoA.
Phosphorylation by pyruvate dehydrogenase kinase, (on E1) switches off the
activity of the complex
dephosphorylation (by a pyruvate dehydrogenase phosphatase) restores
activity of the complex
The phosphatase is activated by Ca2+ and Mg2+
Since ATP binds Mg2+ tighter than ADP does, the concentration of free Mg2+
reflects the ATP/ADP ratio within the mitochondrion
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.
Phosphorylation by pyruvate dehydrogenase kinase, (on E1) switches off the
activity of the complex
dephosphorylation (by a pyruvate dehydrogenase phosphatase) restores
activity of the complex
Insulin accelerates the conversion of pyruvate into acetyl-CoA by stimulating
the phosphatase and thereby the dephosphorylation and resultant activation of
the complex)why? to get glucose out of circulation
pyruvate also stimulates the enzyme
(but by inhibiting the kinase)
Pyruvate Dehydrogenase Kinase are activated by NADH & acetyl-CoA,
Kinase activation involves interaction with E2 subunits to sense changes in
oxidation state & acetylation of lipoamide caused by NADH & acetyl-CoA.
During starvation:
inhibition of Pyruvate Dehydrogenase:
this prevents muscle and other tissues from catabolizing glucose
precursors.
Metabolism shifts toward fat utilization.
Available glucose is spared for use by the brain.
No Krebs, NAD+ plenty, glycolysis only
stimulation of Pyruvate Dehydrogenase Complex:
ADP/ pyruvate
ATP activates... kinase
ADP activates... phosphatase
ADP/ pyruvate inhibit kinase that inactivates PDH
inhibition of Pyruvate Dehydrogenase Complex:
ATP, NADH Acetyl-CoA (means fats catabolized)
ATP activates... kinase
NADH competes with NAD+ for binding to E3.
Acetyl CoA competes with CoA for binding to E2
stimulation of Pyruvate Dehydrogenase:
ADP
PDH phosphorylation: by PDH kinase
removal of Pi phosphoprotein phosphatase
both part of PDH complex!
ATP activates... kinase
ADP activates... Phosphatase.....
inhibition of Pyruvate Dehydrogenase:
ATP, NADH Acetyl-CoA (means fats catabolized)
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.
Entry into cycle and rate of cycle strictly
controlled
Pyruvate  Acetly-CoA irreversible in animals
Figure 17.6 Stryer
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.
Transition reaction inputs and outputs from
glucose
Inputs:
2 pyruvate
2 CoA
2 NAD+
Outputs:
2 acetyl CoA O
2 CO+2 HO C CH
Coenzyme A-SH
2 NADH acetic acid
O
Coenzyme A-S
C
acetyl-CoA
CH3 + H2O
Citric Acid Cycle/Krebs Cycle
citric acid cycle is used to harvest high energy electrons from
carbon fuel.
the central metabolic hub of the cell
produces intermediates which are precursors for fatty acids,
amino acids, nucleotide bases, and cholesterol
named after Hans Krebs who was largely responsible for
elucidating its pathways in the 1930s.
What Are the Metabolic Fates of NADH and
Pyruvate Produced in Glycolysis?
NADH is energy - two possible fates:
If O2 is available:
NADH is re-oxidized in the electron transport pathway, making
ATP in oxidative phosphorylation
If O2 is not available (anaerobic conditions):
NADH is re-oxidized by lactate dehydrogenase (LDH),
providing additional NAD+ for more glycolysis
What Are the Metabolic Fates of NADH and
Pyruvate Produced in Glycolysis?
Pyruvate is also energy - two possible fates:
If O2 is available
pyruvate enters the mitochondria, where it undergoes further
breakdown
If O2 is not available (anaerobic conditions) fermentation
occurs and pyruvate undergoes reduction
Fermentation is an anaeorbic process and does not require
oxygen.
In humans, pyruvate is reduced to lactic acid during
fermentation.