Transcript Pyruvate Dehydrogenase Complex (PDC)

Lecture 25
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Quiz Monday Pentose Phosphate Pathway
This lecture is for WED.
Quiz Friday on TCA cycle
Pyruvate Dehydrogenase Complex (PDC)
3rd stage: carbon-carbon bond cleavage and
formation reactions
• Conversion of three C5 sugars to two C6 sugars and
one C3 (GAP)
• Catalyzed by two enzymes, transaldolase and
transketolase
• Mechanisms generate a stabilized carbanion which
interacts with the electrophilic aldehyde center
Transketolase
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1.
2.
3.
4.
Transketolase catalyzes the transfer of C2 unit from Xu5P to R5P
resulting in GAP and sedoheptulose-7-phosphate (S7P).
Reaction involves a covalent adduct intermediate between Xu5P and
TPP.
Has a thiamine pyrophosphate cofactor that stabilizes the carbanion
formed on cleavage of the C2-C3 bond of Xu5P.
The TPP ylid attacks the carbonyl group of Xu5P (C2)
C2-C3 bond cleavage results in GAP and enzyme bound 2-(1,2dihydroxyethyl)-TPP (resonance stabilized carbanion)
The C2 carbanion attacks the aldehyde carbon of R5P forming an
S7P-TPP adduct.
TPP is eliminated yielding S7P and the regenerated enzyme.
Thiamine Pyrophosphate (B1)
very acidic H since
the electrons can
delocalize into
heteroatoms.
H
CH2
N
CH3
N
+
N
S
CH3
CH2CH2O-P-P
Thiazolium ring
Involved in both oxidative and non-oxidative
decarboxylation as a carrier of "active" aldehydes.
Covalent adduct
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Carbanion intermediate
Transketolase
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Similar to pyruvate decarboxylase mechanism.
Septulose-7-phosphate (S7P) is the the substrate
for transaldolase.
In a second reaction, a C2 unit is transferred from
a second molecule of Xu5P to E4P (product of
transaldolase reaction) to form a molecule of F6P
Transaldolase
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Transfers a C3 unit from S7P to GAP yielding erythrose-4phosphate (E4P) and F6P.
Reactions occurs by aldol cleavage.
S7P forms a Schiff base with an -amino group of Lys from
the enzyme and carbonyl group of S7P.
Transaldolase and Class I aldolase share a common
reaction mechanism.
Both enzymes are  barrel proteins but differ in where
the Lys that forms the Schiff base is located.
•
Essential Lys residue forms a Schiff
base with S7P carbonyl group
•
A Schiff base-stabilized C3 carbanion
is formed in aldol cleavage reaction
between C3-C4 yielding E4P.
The enzyme-bound resonancestabilized carbanion adds to the
carbonyl C of GAP to form F6P.
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The Schiff base hydrolyzes to
regenerate the original enzyme and
release F6P
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Figure 23-31 Summary of carbon skeleton
rearrangements in the pentose phosphate
pathway.
Control of Pentose Phosphate Pathway
1. Principle products are R5P and NADPH.
2. Transaldolase and transketolase convert excess R5P
into glycolytic intermediates when NADPH needs are
higher than the need for nucleotide biosynthesis.
3. GAP and F6P can be consumed through glycolysis and
oxidative phosphorylation.
4. Can also be used for gluconeogenesis to form G6P
5. 1 molecule of G6P can be converted via 6 cycles of
PPP and gluconeogenesis to 6 CO2 molecules and
generate 12 NADPH molecules.
6. Flux through PPP (rate of NADPH production) is
controlled by the glucose-6-phosphate dehydrogense
reaction.
7. G6PDH catalyzes the first committed step of the PPP.
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Pyruvate Dehydrogenase Complex (PDC)
• In aerobic respiration, NAD+ is recycled by the
electron transport chain.
• Also able to utilize energy previously stored as lactate.
• Acetyl-CoA is made from pyruvate through oxidative
decarboxylation by a multienzyme complex,
pyruvate dehydrogense.
• The general reaction catalyzed:
O
C-OH-C=O
CH3
NAD+
+
+
CoA-SH
Acetyl-CoA + NADH + CO2
Gº’ = -8 kcal/mol
Pyruvate Dehydrogenase Complex (PDC)
• Pyruvate dehydrogenase multienzyme complex (PDC)
consists of three enzymes.
• Pyruvate dehydrogenase (E1) form dimers that associate with
E2 at the center of the cubic edges.
• Dihydrolipoyl transacetylase (E2) core of the enzyme. In E.
coli has 24 identical subunits with cubic symmetry.
• Dihydrolipoyl dehydrogenase (E3) form dimers that are
located on the centers of the cube’s six faces.
Gram-negative bacteria have this type.
Another type is dodecahedral form found in eukaryotes and
gram-positive bacteria.
Figure 21-4
Structural organization of
the E. coli PDC.
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Orange spheres are the 24
pyruvate dehydrogenase (E1)
form dimers
Dihydrolipoyl
transacetylase (E2)
core
Dihydrolipoyl
transacetylase (E2)
core indicated by
shaded cube
Purple spheres are the 12
dihydrolipoyl dehydrogenase
(E3) subunits also form
dimers
Combined a and b
Pyruvate Dehydrogenase Complex (PDC)
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5 coenzymes
Thiamine pyrophosphate (TPP)
Flavin adenine dinucleotide (FAD)
Coenzyme A (CoA)
Nicotinamide adenine dicleotide (NAD)
Lipoic acid
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1.
2.
3.
vitamin
thiamine
riboflavin
pantothenic acid
niacin
Multienzyme complexes are catalytically efficient and offer advantages over
separate enzymes
Enzymatic reaction rates are limited by frequency at which enzymes collide with
substrates. In a multi-enzyme complex, the distance the substrates must travel is
minimized, enhancing rates.
Complex formation provides a way of channeling (passing) intermediates between
successive enzymes (minimizes side reactions).
The reactions may be coordinately controlled.
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Figure 21-6
The five reactions of the
PDC.
Pyruvate Dehydrogenase Complex (PDC)
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1.
2.
Acetyl-CoA formation occurs over 5 reactions
Pyruvate dehydrogenase (E1)-decarboxylates pyruvate
using TPP with the intermediate formation of hydroxyethylTPP (like pyruvate decarboxylase).
Dihydrolipoyl transacetylase (E2)-accepts the hydroxyethyl
group from E1.
Thiamine Pyrophosphate (B1)
very acidic H since
the electrons can
delocalize into
heteroatoms.
H
CH2
N
CH3
N
+
N
S
CH3
CH2CH2O-P-P
Thiazolium ring
Involved in both oxidative and non-oxidative
decarboxylation as a carrier of "active" aldehydes.
Mechanism of E1 using TPP
1.
Nucleophilic attack by the dipolar cation (ylid) form of TPP
on the carbonyl carbon of pyruvate to form a covalent
adduct.
2.
Loss of carbon dioxide to generate the carbanion adduct in
which the thiazolium ring of TPP acts as an electron sink.
Pass to next enzyme.
3.
Reaction 1: Pyruvate dehydrogenase (E1) -note how similar to
pyruvate decarboxylase
R
CH3
O
N(+)
(-)
CH2
CH2
S
E1
C-OC=O
CH3
pyruvate
H+
R
CH3
P-P-O
TPP (ylid form)
CO2
CH2
CH2
P-P-O
O
N(+)
S
E1
C-O-
C-OH
CH3
Reaction 2: Dihydrolipoyl transacetylase (E2)
R
CH3
CH2
CH2
N+
C-OH
S
E1
P-P-O
Hydroxyethyl TPP
(HETPP)-E1 complex
H+
S
CH3
S
E2
Lipoamide-E2
Hydroxyethyl group
carbanion attacks the
lipoamide disulfide
causing the reduction
of the disulfide bond
Dihydrolipoyl transacetylase (E2)
R
CH3
N+
S
H+
C-O-H
CH2
CH2
S
E1
P-P-O
Hydroxyethyl TPP
(HETPP)-E1 complex
CH3
HS
E2
The TPP is
eliminated to form
acetyldihydrolipoamide and
regenerate E1
Dihydrolipoyl transacetylase (E2)
R
CH3
CH3 O
C
N+
-
CH2
CH2
S
E1
S
HS
E2
P-P-O
TPP-E1 complex
Back to reaction 1
Acetyldilipoamide-E2
Reaction 3: Dihydrolipoyl transacetylase (E2)
O
CH3 O
C
CoA-S
+
C
S
HS
HS
HS
CH3
CoA-SH
E2
Acetyldilipoamide-E2
E2
dihydrolipamide-E2
E2 catalyzes the transfer of the acetyl group to CoA via a transesterification
reaction where the sulfhydryl group of CoA attacks the acetyl group of the
acetyl dilipoamide-E2 complex.
Reaction 4: Dihydrolipoyl dehydrogenase (E3)
FAD
FAD
S
SH
S
SH
E3 reduced +
E3 oxidized +
S
HS
S
HS
E2
E2
E3 is oxidized and catalyzes the oxidation of dihydrolipoamide completing
the cycle of E2.
Reaction 5: Dihydrolipoyl dehydrogenase (E3)
FAD
FADH2
FAD
SH
S
S
SH
S
S
NAD+
NADH
+ H+
E3 oxidized
E3 is oxidized by the enzyme bound FAD which is reduced to FADH2. This
reduces NAD+ to produce NADH.
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Figure 21-6
The five reactions of the
PDC.
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Figure 21-7
Interconversion of lipoamide
and dihydrolipoamide.
Structure of E2
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Consists of several domains
N-terminal Lipoyl domain (80 residues each)-covalently
binds lipoamide
Peripheral subunit-binding domain (35 residues) binds to
E1 and E3
C-terminal catalytic domain (250 residues) catalytic center
and intersubunit binding.
Linked by 20-40 residue Pro/Ala rich segments.
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Figure 21-8 Domain structure of the dihydrolipoyl
transacetylase (E2) subunit of the PDC.
The number of lipoyl domains depends on the species
E. coli, A. vinelandii, n= 3
Mammals, n = 2
Yeast, n = 1
Figure 21-9 X-Ray structure of a trimer of A. vinelandii
dihydrolipoyl transacetylase (E2) catalytic domains. 24
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The N
terminal
“elbow”
extends over
neighboring
subunit
The CoA and
lipoamide
bound to
enzyme
Structure of E1
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Related to -ketoglutarate dehydrogenase complex and to
branched chain keto acid dehydrogenase complex.
Catalyze the NAD+ linked oxidative decarboxylation of an  keto acid with the transfer of the acyl group to CoA.
No structure of E1 from PDC has been determined but they
make inferences E1 subunits of another keto acid
dehydrogenase (P. putida branched-chain-keto acid
dehydrogenase, a 2-fold symmetric  heterotetramer).
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Figure 21-12a
X-Ray structure of E1 from P.
putida branched-chain -keto acid dehydrogenase. (a)
The 22 heterotetrameric protein.
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Figure 21-12b
X-Ray structure of E1 from P.
putida branched-chain -keto acid dehydrogenase. (b)
A surface diagram of the active site region.
Structure of E3
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The reaction is more complex than depicted.
Contains a redox-active disulfide bond that can form a dithiol.
Catalytic mechanism is similar to glutathione reductase.
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Figure 21-13a
X-Ray structure of dihydrolipoamide
dehydrogenase (E3) from P. putida in complex with FAD and NAD+.
(a) The homodimeric enzyme.
Figure 21-13b
X-Ray structure of dihydrolipoamide
dehydrogenase (E3) from P. putida in complex with FAD and NAD+.
(b) The enzyme’s active site region.
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Redox active disulfide bridge
Mechanism of E3
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The oxidized enzyme E, which contains the redox-active
diulfide bond (S43-S48) binds dihydrolipoamide to form an ES
complex.
His helps with general acid catalysis.
Tyrosine blocks oxidation of FAD by O2 but allows NAD+
access.
Substrate
binding
•Redox disulfide is
reformed.
43
48
•Nucelophillic attack
•Proton abstraction
yields thiolate ions
•NAD+ is reduced to
NADH
•NAD+ binds and the
Tyr is pushed aside.
•Substrate thiolate
displaces S43 with
His as acid catalyst
•S48 forms charge
transfer complex
with FAD
•Lipoamide is
released
•Tyr blocks access
to FAD.
•Charge transfer complex-covalent
bond formed between Cys48 thiolate
and flavin ring. N5 acquires a proton
from Cys43.
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•Cys43 thiolate nucleophillcially attacks
S48 to form the redox active disulfide
bond.
•Release of the FADH- anion.
Regulation of PDC
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PDC regulates the entrance of acetyl units derived from
carbohydrates into the citric acid cycle.
The decarboxylation reaction (E1) is irreversible and it is the only
pathway for acetyl-CoA synthesis from pyruvate in mammals.
2 regulatory systems
Product inhibition by NADH and acetyl-CoA
Covalent modification by phosphorylation/dephosphorylation of the
E1 subunit of pyruvate dehydrogenase.
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Figure 21-17a
Factors controlling the activity of the PDC.
(a) Product inhibition.
Product inhibition
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NADH and acetyl-CoA compete with NAD+ and CoA for
binding sites.
NADH and acetyl-CoA drive reversible transacetylase
(E2) and didhydrolipoyl dehydrogenase (E3) reactions
backwards.
Factors controlling the
activity of the PDC.
(b) Covalent modification in the eukaryotic complex.
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Figure 21-17b
Control by phosporylation/dephosphorylation
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Occurs only in eukaryotic complexes
The E2 subunit has both a pyruvate dehydrogenase
kinase and pyruvate dehydrogenase phosphatase
that act to regulate the E1 subunit.
Kinase inactivates the E1 subunit. Phosphatase
activates the subunit.
Ca2+ is an important secondary messenger, it enhances
phosphatase activity.
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Citric acid cycle: 8 enzymes
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1.
2.
3.
4.
5.
6.
7.
8.
Oxidize an acetyl group to 2 CO2 molecules and generates 3 NADH, 1
FADH2, and 1 GTP.
Citrate synthase: catalyzes the condensation of acetyl-CoA and
oxaloacetate to yield citrate.
Aconitase: isomerizes citrate to the easily oxidized isocitrate.
Isocitrate dehydrogenase: oxidizes isocitrate to the -keto acid
oxalosuccinate, coupled to NADH formation. Oxalosuccinate is then
decarboxylated to form -ketoglutarate. (1st NADH and CO2).
-ketoglutarate dehydrogenase: oxidatively decarboxylates ketoglutarate to succinyl-CoA. (2nd NADH and CO2).
Succinyl-CoA synthetase converts succinyl-CoA to succinate. Forms
GTP.
Succinate dehydrogenase: catalyzes the oxidation of central single bond
of succinate to a trans double bond, yielding fumarate and FADH2.
Fumarase: catalyzes the hydration of the double bond to produce malate.
Malate dehydrogenase: reforms OAA by oxidizing 2ndary OH group to
ketone (3rd NADH)
Total (PDH and TCA)
NAD+
+ pyruvate + CoA
PDH
NADH + acetyl-CoA + CO2
3NAD+ + FAD + GDP + Pi + TCA
acetyl-CoA
Pyruvate
4NAD+
FAD
GDP + Pi
3CO2
4NADH
FADH2
GTP
3NADH + FADH2 + GTP +
CoA + 2CO2
NADH DH
Complex II
Nucleoside
diphosphokinase
12ATP
2ATP
1ATP