The tricarboxylic acid (TCA) cycle
Biochemistry, 4th edition, RH Garrett & CM Grisham,
Brooks/Cole (Cengage); Boston, MA: 2010
pp 563-591
Instructor: Kirill Popov
1. Metabolic sources of acetyl-CoA
2. Enzymes of the Citric Acid Cycle
3. Regulation of the Citric Acid Cycle
4. The amphibolic nature of the Citric Acid Cycle
Stages of cellular respiration
Amino Fatty
acids acids
Stage 1
Acetyl-CoA
production
Glucose
Glycolysis
ee-
e-
pyruvate
dehydrogenase
complex
CO2
e-
Acetyl-CoA
Stage 2
Acetyl-CoA
oxidation
Citrate
Oxaloacetate
e-
Citric
acid cycle
e-
ee-
CO2
CO2
NADH,
FADH2
(reduced e - carriers)
e-
Respiratory
(electron transfer)
chain
ADP + Pi
ATP
2H+ + 1/2O2
H2O
Stage 3
Electron transfer
and oxidative
phosphorylation
Compartmentalization of glycolysis, the citric acid cycle,
and oxidative phosphorylation
Glucose
Glucose
ATP
NADH
Glycolysis
in the cytosol
ATP
Acetyl-CoA
NADH
NADH
ATP
ATP
ATP
ATP
Citric
acid
cycle
Citric acid
cycle and
oxidative
phosphorylation in the
mitochondria
Pyruvate
NADH
ADP
+ P
NAD +
ATP
H2O
O2
CO2
Glycolysis is a preparatory pathway for aerobic
metabolism of glucose
glycolysis
D-Glucose
PDH
2 pyruvate
2 L-lactate
No O2 requirement
for glycolysis
2 acetyl-CoA
2CO2
TCA
4CO2
O2 requirement for pyruvate
dehydrogenase (PDH) plus
TCA cycle activity
Overall reaction catalyzed by pyruvate
dehydrogenase complex
CO2
-
O
O
C
C O
CH3
Pyruvate
+
CoA-SH
NAD+
TPP, lipoate,
FAD
pyruvate dehydrogenase
Complex (E1 + E2 + E3)
NADH
O
S CoA
C
CH3
Acetyl-CoA
ΔG'º = -33.4 kJ/mol
Oxidative decarboxylation of pyruve to acetyl-CoA by the PDH complex
pyruvate
O
CH3
O
C C
-
O
CH3
S
S
E2
C H
S
oxidized
lipoyllysine
FAD
E1
CO2
FAD
E1
S
TPP
1
TPP
OH
E3
E2
E3
O
CH3
C
NADH + H +
S
2
HS
FAD
5
NAD +
TPP
S
S
TPP
E1
E2
oxidized
lipoyllysine
E3
FADH 2 HS
reduced
lipoyllysine
E1
E2
E3
3
CoA-SH
HS
4
O
E1
TPP
E2
CH3
E3
C
S CoA
FAD
acetyl-CoA
Structure of the pyruvate dehydrogenase complex
E1-E3 Outer Shell
E2-E3/BP Inner Shell
Inter Shell Space
L1
L2
E1B
TR Core
L3
E3B
Inner Core
E2
E3BP
Lipoic acid (lipoate) in amide linkage with Lys residue
Oxidized
form
CH2
S
CH2
S
CH
Lipoic
acid
CH2
Reduced
form
O
HS CH2
CH2
HS CH
CH3
C S CH2
CH2
HS CH
CH2
CH2
CH2
CH2
thiazolium
ring
CH2
H
C O
NH2
HN
Lys
residue
of E2
Acetylated
form
CH2 N
N
CH2
CH2
CH3
C
S
O
CH2
CH2
-
O P O P O
CH3
N
O
-
O
O
O
O
-
Thiamine pyrophosphate (TPP)
CH2
H
CH2
CH3
CH
NH
C
NH2
O
Polypeptide chain of
E2 (dihydrolipoyl
transacetylase)
CH2 N
N
CH3
C OH
C S
CH2
N
active
aldehyde
CH3
CH2
-
O P O P O
-
O
Hydroxyethyl thiamine pyrophosphate
-
O
Coenzyme A (CoA)
Reactive
thiol group
NH2
Coenzyme A
H
HS CH2
CH2
H
N C CH2
O
β-Mercaptoethylamine
CH2
H CH3
N C C C CH2
O OH CH3
Pantothenic acid
O-
N
O-
O P O P O CH2
O
O
N
5'
4'
O
N
N
1'
2'
3'
O
OH
Ribose 3'-phosphate
-
O P O
O
CH3
C
S CoA
Acetyl-CoA
Adenine
O-
3’-Phosphoadenosine diphosphate
(3’-P-ADP)
Reactions of the citric acid cycle
Acetyl-CoA
O
CH3
1
C
S CoA
H2O
Condensation
CoA-SH
citrate
synthase
-
O C COO
CH2
8
Dehydrogenation
-
COO
-
HO C COO
-
COO
CH2
COO-
Citrate
Oxaloacetate
malate
dehydrogenase
COO-
Malate
CH2
2a
Dehydration
H2O
aconitase
-
COO
CH2
HO CH
-
C COO
CH2
-
C
COO
cis-Aconitate
COO-
H
7
Hydration
f umarase
H2O
aconitase
NADH
2b
H2O
Hydration
-
COO
CH2
CH
Fumarate
COO
isocitrate
dehydrogenase
succinate
dehydrogenase
Dehydrogenation
α-ketoglutarate CH2 COOdehydrogenase CH
2
complex
-
COO-
CH2
succinyl-CoA
synthetase
CH2
-
COO
CH2
Succinate
Substrate-level
phosphorylation
C
-
COO
5
C S-CoA
GDP
+ Pi
O
Succinyl-CoA
H
CoA-SH
CO2
3
CO2
COO
α-Ketoglutarate
4
Oxidative
decarboxylation
Isocitrate
Oxidative
decarboxylation
O
CH2
CoA-SH
GTP
COO-
HO C
-
6
COO
H C COO
FADH2
CH
-
Formation of citrate
O
CH3
-
O C COO
C
+
S CoA
Acetyl-CoA
CH2
H2O CoA-SH
-
COO
-
-
COO
Oxaloacetate
CH2
HO C COO
citrate
synthase
CH2
COO-
Citrate
ΔG'º = −32.2 kJ/mol
Formation of isocitrate via cis-aconitate
CH2
-
COO
H 2O
CH2
HO C COO
H C
COO-
H
Citrate
-
H 2O
CH2
COO
H C COO
C COO
aconitase
C
COO-
H
cis-Aconitate
-
COO
aconitase
HO C
COO-
H
Isocitrate
ΔG'º = 13.3 kJ/mol
Oxidation of isocitrate to α-ketoglutarate and CO2
CH2
-
COO NAD(P)+ NAD(P)H + H+
H C COO
HO C
-
COO
CH2
-
COO
CH2
isocitrate
dehydrogenase
C
-
COO
H
O
Isocitrate
α-Ketoglutarate
+ CO2
ΔG'º = −20.9 kJ/mol
Oxidation of α-ketoglutarate to succinyl-CoA and CO2
CH2
-
COO
CoA-SH
NAD+
CH2
C
COO-
O
α-Ketoglutarate
NADH
CH2
CH2
α-ketoglutarate
dehydrogenase
complex
-
COO
+ CO2
C S-CoA
O
Succinyl-CoA
ΔG'º = −33.5 kJ/mol
Conversion of succinyl-CoA to succinate
CH2
COO-
-
COO
CH2
C S-CoA
O
Succinyl-CoA
GDP + Pi
GTP
succinyl-CoA
synthetase
CoA-SH
CH2
CH2
-
COO
Succinate
ΔG'º = −2.9 kJ/mol
Succiny-CoA synthase reaction
Succinyl-CoA
-
O
O
Succinate
C
CH2
COO-
CH2
CH2
C
O
-
CoA-SH
CH2
O
O
S-CoA
-
C
GDP
COO
GTP
CH2
CH2 His
1
Pi
C
O
His
succinyl-CoA
synthetase
O
2
His
P
P
Enzyme-bound
succinyl
phosphate
Phosphoenzyme
3
His
Oxidation of succinate to fumarate
-
-
COO
COO
FAD
CH2
CH2
-
FADH2
succinate
dehydrogenase
CH
CH
-
COO
COO
Succinate
Fumarate
ΔG'º = 0 kJ/mol
Hydration of fumarate to malate
COO-
-
COO
C H
H C
fumarase
-
COO
Fumarate
HO CH
H2O
H C H
-
COO
L-Malate
ΔG'º = -3.8 kJ/mol
Oxidation of malate to oxaloacetate
-
COO
-
NAD+
NADH + H+
HO CH
O C
H C H
-
COO
L-Malate
COO
CH2
malate
dehydrogenase
-
COO
Oxaloacetate
ΔG'º = 29.7 kJ/mol
Products of one turn of the citric acid cycle
Acetyl-CoA
Citrate
Oxaloacetate
Isocitrate
NADH
NADH
CO2
Malate
α-Ketoglutarate
CO2
Fumarate
NADH
Succinyl-CoA
FADH 2
Succinate
GTP
Stoichiometry of Coenzyme Reduction and ATP Formation in the Aerobic Oxidation of Glucose via Glycolysis,
Pyruvate Dehydrogenase Reaction, the Citric Acid Cycle, and Oxidative Phosphorylation
Reaction
Number of ATP or
reduced
coenzymes
directly formed
Number of
ATP
ultimately
formed*
Glucose → glucose 6-phosphate
-1 ATP
-1
Fructose 6-phosphate → fructose 1,6-bisphosphate
-1 ATP
-1
2 NADH
3-5
2 1,3-Bisphosphoglycerate → 2 3-phosphoglycerate
2 ATP
2
2 Phosphoenolpyruvate → 2 pyruvate
2 ATP
2
2 Pyruvate → 2 acetyl-CoA
2 NADH
5
2 Isocitrate → 2 α-ketoglutarate
2 NADH
5
2 α-Ketoglutarate → 2 succinyl-CoA
2 NAD
5
2 Succinyl-CoA → 2 succinate
2 GTP
2
2 Succinate → 2 fumarate
2 FADH 2
3
2 Malate → 2 oxaloacetate
2 NADH
5
2 Glyceraldehyde 3-phosphate → 2 1,3-bisphosphoglycerate
Total
*This is calculated as 2.5 ATP per NADH and 1.5 ATP per FADH2.
A negative value indicates consumption.
30-32
Role of the citric acid cycle in anabolism
pyruvate
Fatty acids,
sterols
Glucose
pyruvate
carboxylase
Acetyl-CoA
PEP carboxylase
Phosphoenolpyruvate
(PEP)
Serine
Glycine
Cysteine
Phenylalanine
Tyrosine
Tryptophan
Oxaloacetate
Citrate
PEP
carboxylase
Aspartate
Asparagine
Malate
α-Ketoglutarate
Glutamine
Proline
Arginine
Glutamate
malic
enzyme
Purines
Pyrimidines
pyruvate
Succinyl-CoA
Porphyrins,
heme
Anaplerotic Reactions
Reaction
Tissue(s)/organism(s)
pyruvate carboxylase
Pyruvate + HCO3- + ATP
oxaloacetate + ADP + Pi
Liver, kidney
PEP carboxykinase
Phosphoenolpyruvate + CO2 + GDP
oxaloacetate + GTP
Heart, skeletal muscle
PEP carboxylase
oxaloacetate + Pi
Phosphoenolpyruvate + HCO3-
Plants, yeast, bacteria
malic enzyme
Pyruvate +
HCO3-
+ NAD(P)H
+
Malate + NAD(P)
Eukaryotes and
prokaryotes
The role of biotin in pyruvate carboxylase reaction
Step 1
H
O
-
O C OH
ATP
O
Bicarbonate
ADP
C
O
O
O
HO P O C
O
NH
Carbon
dioxide S
NH
C N
-
(CH2)3
O
CH2
(CH2 )3
CH2
S
C O
Pi
O H
-
O
N
O
O
C O
NH
NH
Lys
Lys
pyruvate
carboxylase
OH
O
O
O
O
C C CH2
-
Oxaloacetate
OH
S
NH
C N
-
+
C
O
NH
N
O
(CH2)3
CH2
O
-
O
O
(CH2 )3
S
C C CH2
C O
-
NH
O
Lys
O
-
Lys
O
-
H+
O
keto form
O
O H
C C C H
-
Step 2
C O
NH
enol form
C C CH2
CH2
O
H
pyruvate
Regulation of PDH complex
Product inhibition
FAD
NAD+
SH
SH
5
NADH
E3
CO2
FAD
Lipoamide
HydroxyethylTPP
4
S
S
1
E1
E2
2
Dihydrolipoamide
Acetyldihydrolipoamide
Pyruvate
TPP
3
Acetyl-CoA
CoA
Covalent modification
Pi
E1−OH (active)
pyruvate
dehydrogenase
phosphatase
H 2O
ATP
pyruvate
dehydrogenase
kinase
E1−OPO32− (inactive)
ADP
Regulation of metabolite flow through the citric acid cycle
Pyruvate
ATP, acetyl-CoA,
NADH, fatty acids
pyruvate
dehydrogenase
complex
AMP, CoA, NAD+, Ca2+
Acetyl-CoA
NADH, succinyl-CoA, citrate, ATP
ADP
citrate
synthase
Citrate
Oxaloacetate
Isocitrate
malate
dehydrogenase
ATP
isocitrate
dehydrogenase
Ca2+, ADP
NADH
Malate
FADH2
α-Ketoglutarate
α-ketoglutarate
dehydrogenase
succinyl-CoA, NADH
Fumarate
Ca2+
succinate
dehydrogenase
Succinyl-CoA
Succinate
GTP
ATP
1.
Pyruvate, the product of glycolysis, is converted to acetyl-CoA, the starting
material for the citric acid cycle, by the pyruvate dehydrogenase multienzyme
complex
2.
The citric acid cycle is a central catabolic pathway in which compounds
derived from the breakdown of carbohydrates, fats and proteins are oxidized
to CO2, with most of the energy of oxidation temporarily held in the electron
carriers FADH2 and NADH
3.
Acetyl-CoA enters the citric acid cycle through its condensation with
oxaloacetate to form citrate; in seven sequential reactions, the citric acid
cycle converts citrate to oxaloacetate and releases two CO2; for each acetylCoA oxidized, the energy gain consists of three molecules of NADH, one
FADH2 and one GTP
4.
The citric acid cycle is amphibolic, serving in both catabolism and anabolism
5.
The overall rate of the citric acid cycle is controlled by the rate of conversion
of pyruvate to acetyl-CoA and by the flux through citrate synthase, isocitrate
dehydrogenase, and α-ketoglutarate dehydrogenase