Lecture 26
– TCA cycle
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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)
Citric acid cycle
3NAD+ + FAD + GDP + Pi + acetyl-CoA
3NADH + FADH2 + GTP + CoA + 2CO2
3NADH + FADH2 are oxidized by the electron transport
chain and drive ATP synthesis.
Citrate synthase: reaction 1
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Catalyzes the condensation of acetyl-CoA and oxaloacetate to form citrate.
Oxaloacetate has to bind to the enzyme before acetyl-CoA.
Oxaloacetate binds to the enzyme causing a conformational shift that opens the
acetyl-CoA binding site. (induced fit)
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Reaction mechanism is a mixed aldol-Claisen ester condensation (acid-base
catalysis).
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Acetyl forms an enol intermediate.
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Three important amino acids: His274, Asp375, His320
1.
The formation of enolate form of acetyl-CoA is the rate-limiting step. Asp375 acts as
a general base to remove a proton from the methyl group of the acetyl-CoA. His 274
is hydrogen bonded to acetyl-CoA.
2.
Citryl-CoA is formed in a second acid-base catalyzed reaction step. Acetyl-CoA
enolate form attacks oxaloacetate.
3.
Citryl-CoA is hydrolyzed to citrate and CoA.
Stereospecific reactions (acetate onlly forms citrate’s pro-S carboxymethyl group.
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Aconitase: reaction 2
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Catalyzes the reversible isomerization of citrate and
isocitrate with cis-aconitate as an intermediate.
Citrate is prochiral so aconitase can distinguish between
citrate’s pro-R and pro-S carboxymethyl groups.
Has a covalently bound [4Fe-4S] iron-sulfur cluster.
Fea atom coordinates with the OH group of citrate
The iron-sulfur cluster does not perform a redox reaction
but instead is able to stabilize the ligand-substrate
complex.
Second stage of the reaction rehydrates cis-aconitate’s
double bond in a stereospecific trans addition to form
only the 2R,3S isocitrate form.
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Isocitrate dehydrogenase: reaction 3
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Catalyzes the oxidiation of isocitrate to form aketoglutarate
1st reaction to produce NADH and CO2.
Activated by AMP and ADP
Inhibited by NADH and NADPH
Competitively bind to the NAD+ binding site.
Requires Mn2+ or Mg2+ cofactor.
Mechanistically-oxidize to the b-keto acid.
2 forms of the enzyme
Mitochondrial form is NAD+ dependant [ADP]
E. coli, mitochondrial, cytoplasmic forms NADP+
dependant.
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Figure 21-21 Probable reaction
mechanism of isocitrate dehydrogenase.
-ketoglutarte dehydrogenase complex
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Catalyzes the oxidiation and decarboxylation of ketoglutarate to produce succinyl-CoA.
Consists of -ketoglutarte dehydrogenase (E1),
dihydrolipoyl transsuccinylase (E2), and
dihydrolipoyl dehydrogenase (E3).
Mechanistically resembles PDC.
2nd reaction to produce NADH and CO2.
5 coenzymes (TPP, lipoic acid, CoA, FAD, NAD+)
Product inhibition (Succinyl-CoA), NADH
Reaction 1: -ketoglutarate dehydrogenase
R
-ketoglutarate
O
(+)
N
CH3
C
O
(-)
C=O
CH2
CH2
S
E1
P-P-O
TPP (ylid form)
CH2
CH2
R
H+
CH3
O
N(+)
C-O-
C-OH
CH2
C-OO
CO2
CH2
CH2
P-P-O
S
E1
CH2
C-OO
Reaction 2: Dihydrolipoyl transacetylase (E2)
R
CH3
N+
C-OH
H+
S
CH2
CH2
CH2
P-P-O
S
E1 CH2
S
C-OO
-hydroxy--carboxypropyl TPP-E1
complex
E2
Lipoamide-E2
-hydroxy group
carbanion attacks the
lipoamide disulfide
causing the reduction
of the disulfide bond
Dihydrolipoyl transacetylase (E2)
R
CH3
S
N+
H+
C-O-H
CH2
CH2
CH2
P-P-O
S
E1 CH2
HS
E2
C-OO
-hydroxy--carboxypropyl TPP-E1
complex
The TPP is
eliminated to form
succinyl dihydrolipoamide and
regenerate E1
Dihydrolipoyl transacetylase (E2)
R
CH3
O
N+
-
CH2
CH2
S
E1
C-O-
CH2
CH2 O
C
S
P-P-O
TPP-E1 complex
Back to reaction 1
HS
E2
Succinyldilipoamide-E2
Reaction
transacetylase (E2)
O 3: Dihydrolipoyl
C-OO
CH2
CoA-S C CH2-CH2-COOCH2 O
Succinyl-CoA
C
+
S
HS
CoA-SH
HS
HS
E2
Succinyldilipoamide-E2
E2
dihydrolipamide-E2
E2 catalyzes the transfer of the succinyl 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.
Succinyl-CoA Synthetase
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Hydrolyzes the “high-energy” succinyl-CoA with
the coupled synthesis of a “high-energy”
nucleoside triphosphate.
In mammals, GTP
In bacteria and plants, ATP.
Succinyl-CoA Synthetase
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Mechanistically:
Succinyl-P
Pi
CoASH
Enz-His
Succinyl-CoA
Succinate
Enz-His
Enz-His-P
GTP
Mg++
GDP
Enz-His-P
Succinate
Succinyl-CoA Synthetase
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Mechanistically:
Pi
CoASH
Succinyl-P
Enz-His
Succinyl-CoA
Succinate
Enz-His
Enz-His-P
GTP
Mg++
GDP
Enz-His-P
Succinate
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Figure 21-22a
Reactions catalyzed by
succinyl-CoA synthetase. Formation of succinyl
phosphate, a “high-energy” mixed anhydride.
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Figure 21-22b
Reactions catalyzed by
succinyl-CoA synthetase. Formation of phosphoryl–His,
a “high-energy” intermediate.
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Figure 21-22c
Reactions catalyzed by
succinyl-CoA synthetase. Transfer of the phosphoryl
group to GDP, forming GTP.
Succinate dehydrogenase
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Only makes the trans-fumarate.
Donates electrons directly into complex II of the
respiratory chain (ubiquinone (Q)).
If the respiratory chain is inhibited, FAD is
unable to accept electrons and TCA cycle stops.
Inhibited by OAA, activated by coenzyme Q (part
of electron tranport chain).
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Figure 21-23 Covalent attachment of FAD to a His
residue of succinate dehydrogenase.
Succinate dehydrogenase
Electron
transport chain
COOH-C-H
H-C-H
COOSuccinate
FAD
FADH2
H-C-COO-OOC-C-H
Succinate
dehydrogenase
Fumarate
Succinate dehydrogenase
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Catalyzes the stereospecific dehydrogenation of
succinate to fumurate.
Enzyme strongly inhibited by malonate
(competitive inhibitor).
Contains an FAD-electron acceptor.
FAD functions to oxidize alkanes to alkenes (vs.
NAD+ which oxidizes alcohols to aldehydes and
ketones).
FAD covalently linked to His from enzyme.
Fumarase (fumarate hydratase)
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Catalyzes the stereospecific dehydrogenation of
succinate to fumurate.
Only catalyzes the trans-fumarate
Competitively inhibited by maleate (cis doublebond).
Fumarase
H-C-COO- H O
2
-OOC-C-H
Fumarase
Fumarate
COOHO-C-H
H-C-H
COOS-malate
Malate dehydrogenase
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Catalyzes the final reaction of the citric acid cycle-regeneration of
oxaloacetate.
Oxidizes S-malate’s OH group to a ketone in an NAD+ dependent
reaction.
Produces NADH.
COOHO-C-H
H-C-H
COOS-malate
NAD+
NADH
Malate
dehydrogenase
COOO=C-H
H-C-H
COOOxaloacetate
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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
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Figure 21-25 Regulation of the citric acid cycle.
Regulation of citric acid cycle
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Rate-controlling enzymes: citrate synthase, isocitrate
dehydrogenase, -ketoglutarate dehydrogenase.
Regulated by substrate availability, product inhibition and inhibition
by other cycle intermediates (generally simpler than glycolysis).
Citrate synthase- inhibited by citrate, -KG, succ-CoA, NADH,
activated by OAA and CoASH.
Isocitrate dehydrogenase-Requires AMP/ADP Activated by Ca2+,
inhibited by NADPH or NADH
-ketoglutarate dehydrogenase-inhibited by Succ-CoA, NADH,
ATP. Activated by Ca2+
Pyruvate dehydrogenase-inhibited by NADH and acetyl-CoA
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Figure 21-26 Amphibolic functions of the citric acid
cycle.
Pathways that use citric acid cycle intermediates
Reactions that utilize intermediates of TCA cycle are called cataplerotic
reactions
1.
Gluconeogenesis-in cytosol uses OAA. In the mitochondria uses
malate (transported across the membrane).
2.
Lipid biosynthesis-requires acetyl-CoA. Transported across the
membrane by the breakdown of citrate.
ATP + citrate + CoA
3.
ADP + Pi + oxaloacetate + acetyl-CoA
Amino acid biosynthesis-can use -ketoglutarate to form glutamic
acid in a reductive amination reaction (uses NAD+ or NADP+
depending on enzyme)
-ketoglutarate + NAD(P)H + NH4+
glutamate + NAD(P)+ + H2O
Pathways that use citric acid cycle intermediates
3.
Amino acid biosynthesis-can also use -ketoglutarate
and oxaloacactate in transamination reactions
-ketoglutarate + alanine
oxaloacetate + alanine
4.
5.
glutamate + pyruvate
aspartate + pyruvate
Porphyrin biosynthesis- utilizes succinyl-CoA
Complete oxidation of amino acids - amino acids first
converted to PE by PEPCK
Pathways that make citric acid cycle intermediates
Reactions that replenish intermediates of TCA cycle are called
anaplerotic reactions
Pyruvate carboxylase- produces oxaloacetate
Pyruvate + CO2 + ATP + H2O
oxaloacetate + ADP + Pi
Degradative pathways generate TCA cycle intermediates
1.
Oxidation of odd-chain fatty acids generates succinyl-CoA
2.
Ile, Met, Val generate succinyl-CoA
3.
Transamination and deamination of amino acids leads to ketoglutarate and oxaloacetate.
Each NADH yields ≈ 3ATP
Each FADH2 yields ≈ 2ATP
Total yields ≈ 38ATP for each
fully oxidized glucose.
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Glyoxylate cycle
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The glyoxylate cycle results in the net conversion of two acetyl-CoA
to succinate instead of 4 CO2 in citric acid cycle.
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Succinate is transferred to mitochondrion where it can be converted
to OAA (TCA)
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Can go to cytosol where it is converted to oxaloacetate for
gluconeogenesis.
Net reaction
2Ac-CoA + 2NAD+ + FAD
OAA + 2CoA + 2NADH +FADH2 + 2H+
Plants are able to convert fatty acids to glucose through this
pathway
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