• In determining the fate of pyruvate after glycolysis, two
questions need to be asked;
1. Is there oxygen present i.e. aerobic conditions?
2. Are there any mitochondria present to facilitate TCA
cycle and ETC?
• In aerobic conditions, with mitochondria present, the
two molecules of pyruvate produced from one molecule
of glucose, are then transported to the mitochondria of
the cell where they enter the TCA cycle.
• Via the TCA and ETC, energy is then generated and
NAD+ is regenerated in the ETC.
Milton A Wesuta, Biochemistry-MUST 197
Citric Acid Cycle
• Fate of Pyruvate under aerobic conditions
TriCarboxylic Acid (TCA) cycle
Kreb’s Cycle
• Central Pathway to metabolism
• Carbohydrates, lipids and Proteins are degraded by
oxidation
• Involves (i) A transition stage – conversion of Pyruvate to
give Acetyl CoA and NADH
(ii) Entry and further oxidation to give CO2 (exhaled)
and Reducing equivalents i.e. more NADH and FADH2
Milton A Wesuta, Biochemistry-MUST 198
Entry into the TCA Cycle
Pyruvate is transported there via pyruvate translocase. For each
initial glucose molecule, two pyruvate molecules will enter the
mitochondria
Milton A Wesuta, Biochemistry-MUST 199
CONVERSION OF PYRUVATE
TO ACETYL COA
A. Oxidative decarboxylation of pyruvate
• Conversion of pyruvate to acetyl CoA
• Pyruvate enters mitochondria and converted to
acetyl CoA by the pyruvate dehydrogenase complex
(PDH), which is a multi-enzyme complex.
• Strictly speaking, the pyruvate dehydrogenase
complex is not part of the TCA cycle, but is a major
source of acetyl CoA—the two-carbon
substrate/raw material for the cycle.
200
Oxidative
decarboxylation of
pyruvate. Product
inhibition is shown, but
covalent modification is
the key method of
regulation for PDH. PDH
is active when
. dephosphorylated
201
Pyruvate Dehydrogenase, catalyzes oxidative
decarboxylation of pyruvate, to form acetyl-CoA.
Pyruvate Dehydrogenase
O O HSCoA O
H3C C C O H 3C C S CoA + CO2
pyruvate + acetyl-CoA
NAD NADH
202
Pyruvate Dehydrogenase
Subunits
Enzyme Abbreviated Prosthetic Group
Pyruvate E1 Thiamine pyrophosphate (TPP)
Dehydrogenase
Dihydrolipoyl E2 Lipoamide/ Lipoic acid and
CoA
Transacetylase
Dihydrolipoyl E3 FAD and NAD+
Dehydrogenase
Component enzymes: The pyruvate dehydrogenase complex
(PDH) is a multi-molecular aggregate of three enzymes, pyruvate
dehydrogenase (E1, also called a decarboxylase), dihydrolipoyl
transacetylase (E2), and dihydrolipoyl dehydrogenase (E3). 203
Sequence of reactions
catalyzed by Pyruvate
Dehydrogenase complex:
1. The keto C of pyruvate reacts with the TPP on E1 to yield
Hydroxyethyl-TPP as CO2 is lost. Hydroxyethyl-TPP
remains.
2. The hydroxyethyl group on TPP of E1 reacts with the disulfide
(S-S) of lipoamide on E2 and forms acetyl
dihydrolipoamide. The hydroxyethyl gp (CH3CHOH)
forms the acetyl gp (CH3CO).
3. The acetyl gp is transferred from acetyl dihydrolipoamide
to coenzyme A, yielding acetyl CoA (major product of
PDH enzyme). The lipoamide disulfide is reduced to a dithiol
(SH-SH)
Sequence of reactions
(continued)
4. The reduced lipoamide (dihydrolipoamide), swings
over to the E3 active site.
Dihydrolipoamide is re-oxidized to the disulfide (S-S), as
2 e- + 2 H+ are transferred to a disulfide on E3 (disulfide
interchange).
E2 and E3 have disulfide (S-S) at the active sites.
5. The dithiol (SH-SH) on E3 is re-oxidized as 2 e- + 2H+
are transferred to FAD (forming FADH2)
The resulting FADH2 is re-oxidized by electron transfer
to NAD+, to yield NADH (other major product of
PDH enzyme) + H+.
Pyruvate Dehydrogenase
Milton A Wesuta, Biochemistry-MUST 206
• At the end of the reaction, 2 molecules of pyruvate
form 2 molecules of Acetyl Co-A + 2 molecules of
carbon dioxide + NADH. This reaction is also
IRREVERSIBLE.
• Once Acetyl CoA is formed, it is combined to
oxaloacetate in the matrix of the mitochondria to
form citrate in the TCA cycle.
• This occurs in plant, animal and some microbial cells
with the essential requirement that aerobic
conditions exist and mitochondria are present.
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Citrate Synthase
1) Citrate synthase reaction
Only step in TCA cycle that involves the
formation of a C-C bond
Cycle begins with the combination of acetyl CoA and
oxaloacetate producing 6-C compound Citrate
• Irreversible reaction
• Acetyl CoA + oxaloacetate citrate and CoA
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Milton A Wesuta, Biochemistry-MUST 210
2) Aconitase reaction
• Isomerisation/ re-arrangement step involving two
reactions/steps
• Citrate isocitrate
• Aconitase is inhibited by fluorocitrate, a very toxic
substance
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Aconitase
Milton A Wesuta, Biochemistry-MUST 212
3) isocitrate dehydrogenase rxn
Functions with NAD+ as electron acceptor
• Substrate first oxidized by NAD+ (yielding NADH),
then decarboxylated.
• Isocitrate α-ketoglutarate + CO2 + NADH +
H+
Milton A Wesuta, Biochemistry-MUST 213
Isocitrate Dehydrogenase
Milton A Wesuta, Biochemistry-MUST 214
• α-ketoglutarate dehydrogenase complex
• α-ketoglutarate first decarboxylated, oxidized (by
NAD+ yielding NADH ), and HS-CoA added
• Step produces high energy compound succinyl
CoA
• Enzyme complex similar the PDH, but has
dihydrolipoyl transsuccinylase instead of
dihydrolipoyl transacetylase (E2)
• Inhibited by Arsenate
Milton A Wesuta, Biochemistry-MUST 215
-Ketoglutarate
Dehydrogenase
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Succinyl CoA synthetase or
succinate thiokinase
• succinyl CoA succinate
• Energy from the high energy compound (thioester
bond) is transformed into GTP (or ATP)
• This is substrate level phosphorylation
GDP +Pi GTP mammals
ADP +Pi ATP plants and bacteria
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Succinyl-CoA Synthetase
Milton A Wesuta, Biochemistry-MUST 218
• Enzyme is embedded in inner mitochondrial
membrane.
• Has FAD covalently bound to it (as its prosthetic
group).
• Converts succinate fumarate with
generation of FADH2
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Succinate Fumarate + 2H++ 2e-
Succinate dehydrogenase
CH2COOH CHCOOH
COOH
CH2 CHCOOH
CH2COOH
COOH
Malonate
HO2C-(CH2)3-CO2H HO2C-CH2-CO-CO2H
Inhibitors of succinate Oxaloacetate(OAA)
Glutarate dehydrogenase
Milton A Wesuta P. 221 of 65 2/19/2025
7) Fumarase reaction
•Catalyses addition of H2O across the double
bond in fumarate forming malate
•fumarate malate
2/19/2025 Milton A Wesuta P. 222 of 65
Fumarase
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8) Malate dehydrogenase
•Malate is oxidized by NAD+ to oxaloacetate,
producing another molecule of NADH
•malate oxaloacetate
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Malate Dehydrogenase
Milton A Wesuta, Biochemistry-MUST 225
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Reduced Coenzymes Fuel
ATP Production
• Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi +2 H20 HS-
CoA + 3NADH + FADH2 + GTP + 2 CO2 + 2 H+
• Isocitrate Dehydrogenase 1 NADH=3 ATPs
• a-ketoglutarate dehydrogenase 1 NADH=3 ATPs
• Succinyl-CoA synthetase 1 GTP=1 ATP
• Succinate dehydrogenase 1 FADH2=2 ATPs
• Malate Dehydrogenase 1 NADH=3 ATPs
• Total of 12 ATPs gained from oxidation of 1 Acetyl-CoA
Milton A Wesuta, Biochemistry-MUST 227
Milton A Wesuta, Biochemistry-MUST 228
Regulation of TCA Cycle
229
Milton A Wesuta, Biochemistry-MUST
Regulation of PDH
• Regulates entrance of acetyl units from
carbohydrates into TCA
• E1 activity is irreversible
• Two regulatory systems employed
• (1) direct product inhibition by high [NADH] and
[Acetyl CoA]. These molecules also activate PDH
kinase that converts the active PDH to an inactive
phosphorylated form.
• (2) Covalent modification by dephosphorylation by
phosphoprotein phosophatase
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Regulation of TCA Cycle
• By modulation of key enzymes and substrate
availability
• Depends on continuous supply of NAD+, FAD &
ADP
• Key enzymes are Citrate synthase, Isocitrate
Dehydrogenase and α-ketoglutarate dehydrogenase
are highly regulated because they catalyse steps that
represent important branch points.
• Citrate synthase is stimulated by Acetyl-CoA &
OAA availability, allosterically inhibited by succinyl-
CoA, NADH & ATP levels
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Regulation of TCA Cycle
• Isocitrate DH is stimulated by ADP & NAD+ &
inhibited by ATP & NADH. IDH must be active to
drive cycle forward since Aconitase reaction is
reversible.
• α-KDH is also involved in amino acid synthesis. So
the need for careful regulation. When cell’s energy
stores are low, activation retains α-KD within the
cycle. When NADH rises α-KD is made available for
biosynthetic reactions
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Amphibolic nature of TCA
• Cycle is amphibolic in that it is both catabolic & anabolic
• OAA, α-KD, succinyl-CoA and Acetyl-CoA are precursors
of biosynthetic pathways
• OAA – gluconeogenesis & amino acid biosynthesis
• α-KD – amino acid synthesis
• Succinyl-CoA – heme synthesis
• Acetyl-CoA – Fatty acid & Cholesterol synthesis
• Cycle also degrades Acetyl-CoA from Carbohydrates, fatty
acids and amino acids to form CO2 and energy is
conserved in the reduced coenzyme molecules.
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Protein/amino acid
Catabolites feed
Into the TCA Cycle
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Fatty acids
breakdown and feed
into the TCA Cycle
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TCA Cycle provides
intermediates for
many biosynthetic
processes
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The Anaplerotic Reactions
The "filling up" reactions that increase [cycle intermediates]
drained by anabolic processes
• PEP carboxylase - converts PEP to oxaloacetate
• Pyruvate carboxylase - converts pyruvate to oxaloacetate
• Malic enzyme converts pyruvate into malate
• Formation of α-ketoglutarate & OAA by transaminases
• α-ketoglutarate formation from glutamate by glutamate
dehydrogenase
• Succinyl formation from Ile, Val, Met and Thr
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