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
Acetyl-CoA
CO2
NADH,FADH2
(reduced e- carriers)
ATPADP + Pi
Respiratory(electron transfer)
chain
Citricacid cycle
Glycolysis
Stage 3Electron transfer
and oxidativephosphorylation
Stage 2Acetyl-CoAoxidation
Stage 1Acetyl-CoAproduction
CO2
e-
e-
e-
e-
e-
e-
e- e-
e-
Aminoacids
Fattyacids Glucose
2H+ + 1/2O2
H2O
CO2
pyruvatedehydrogenasecomplex
OxaloacetateCitrate
Stages of cellular respiration
Compartmentalization of glycolysis, the citric acid cycle, and oxidative phosphorylation
ATP
ATP
ATP
P
NADHNADH
NADHNAD+
ADP
H2O
O2 CO2
NADH
Acetyl-CoA
Glucose
Citricacidcycle
Citric acidcycle andoxidativephosphoryla-tion in the mitochondria
Glycolysisin the cytosol
Glucose
Pyruvate
ATP
ATP
ATP
ATP
+
D-Glucose 2 pyruvate 2 acetyl-CoAglycolysis PDH
2CO2 4CO22 L-lactate
No O2 requirementfor glycolysis
O2 requirement for pyruvatedehydrogenase (PDH) plus
TCA cycle activity
TCA
Glycolysis is a preparatory pathway for aerobic metabolism of glucose
C
C
CH3
O-O
OC
CH3
SO CoA
Pyruvate Acetyl-CoA
+CoA-SH
ΔG'º = -33.4 kJ/mol
NADH
pyruvatedehydrogenaseComplex (E1 + E2 + E3)
NAD+TPP, lipoate,
FAD
CO2
Overall reaction catalyzed by pyruvatedehydrogenase complex
CCH3
OH
TPP
H
S
S
CCH3
O
HS
S
HS
HS
CCH3
O
CoAS
S
S
S
S
NADH + H+
FADH2
FAD
NAD+
FAD
FAD
FAD
TPP
TPP
TPP
TPP
1
2
3
4
5
pyruvate
oxidizedlipoyllysine
reducedlipoyllysine
oxidizedlipoyllysine
CO2
acetyl-CoA
CoA-SH
E2E1 E3
E2E1 E3
E2E1 E3
E2E1 E3
E2E1 E3
O-
O
CCCH3
O
Oxidative decarboxylation of pyruve to acetyl-CoA by the PDH complex
Structure of the pyruvate dehydrogenase complex
L1 L2 E1B TR Core
L3 E3B Inner Core E3BPE2
E2-E3/BP Inner ShellE1-E3 Outer Shell
Inter Shell Space
CHCH2
CH2
CH2
S
HS
CCH3
O
Polypeptide chain ofE2 (dihydrolipoyltransacetylase)
Lipoicacid
Acetylatedform
Reducedform
Oxidizedform
Lysresidue
of E2
CHCH2
CH2
CH2
HS
HS
Thiamine pyrophosphate (TPP)
thiazoliumring
activealdehyde
Hydroxyethyl thiamine pyrophosphate
N
N
NH2
CH2
CH3
SN
C
CH3
CH2 CH2 O P O P O-
OO
O- O-
H
N
N
NH2
CH2
CH3
SN
C
CH3
CH2 CH2 O P O P O-
OO
O- O-
C
H
OHCH3
S
S CHCH2
CH2
CH2
CH2
CH2
CH2
C O
HN
CH2
CH2
CH2
CH2
CHNH C
O
Lipoic acid (lipoate) in amide linkage with Lys residue
O
OHO
CH2
N
N
N
N
NH2
P O-
O-
O
OPOPOCH2CCCNCH2CH2CNCH2CH2
H
O
H
O
H CH3
CH3OH
O- O-
O O
HS
Pantothenic acidβ-Mercaptoethylamine
3’-Phosphoadenosine diphosphate(3’-P-ADP)
Coenzyme AAdenine
Ribose 3'-phosphate
Reactivethiol group
1'
2'3'
4'
5'
Acetyl-CoA
CH3 CO
S CoA
Coenzyme A (CoA)
CCH3
O
CoAS
CH2
C
COO-
HO
CH2 COO-
COO-
CH2
C
COO-
C COO-
COO-
H
CH2
C
COO-
C COO-
COO-H
HO
H
CH2
CH2
COO-
C COO-
OCH2
CH2
COO-
C
O
S-CoA
CH2
CH2
COO-
COO-
CH
CH
COO-
COO-
CH
CH2
COO-
COO-
HO
CO
CH2 COO-
COO-
FADH2
NADH
Acetyl-CoA
1
GTP
H2O
H2O
H2O
H2O
GDP+ Pi
CO2
CO2
Malate
Oxaloacetate Citrate
cis-Aconitate
Isocitrate
α-Ketoglutarate
Succinyl-CoA
Fumarate
Succinate
Hydration
Dehydrogenation
Hydration
Oxidativedecarboxylation
Dehydration
Dehydrogenation
Substrate-levelphosphorylation
CoA-SH
Oxidativedecarboxylation
CoA-SH
CoA-SH
fumarase
succinyl-CoAsynthetase
malatedehydrogenase
citratesynthase
aconitase
α-ketoglutaratedehydrogenase
complex
aconitase
isocitratedehydrogenase
succinatedehydrogenase
3
4
5
6
8
7
Condensation
2a
2b
Reactions of the citric acid cycle
CO
CH2 COO-
COO-CH2
C
COO-
HO
CH2 COO-
COO-CCH3
O
CoAS
ΔG'º = −32.2 kJ/mol
citratesynthase
Acetyl-CoA Oxaloacetate Citrate
H2O CoA-SH
+
Formation of citrate
CH2
C
COO-
C COO-
COO-
H
CH2
C
COO-
C COO-
COO-H
HO
H
CH2
C
COO-
HO
C COO-
COO-
H
H
H2O H2O
ΔG'º = 13.3 kJ/mol
aconitase
cis-Aconitate
aconitase
IsocitrateCitrate
Formation of isocitrate via cis-aconitate
CH2
C
COO-
C COO-
COO-H
HO
H
CH2
CH2
COO-
C COO-
O
isocitratedehydrogenase
+ CO2
Isocitrate
ΔG'º = −20.9 kJ/mol
α-Ketoglutarate
NAD(P)H + H+NAD(P)+
Oxidation of isocitrate to α-ketoglutarate and CO2
CH2
CH2
COO-
C COO-
O
CH2
CH2
COO-
C
O
S-CoA
+ CO2
ΔG'º = −33.5 kJ/mol
α-Ketoglutarate
CoA-SH NAD+
Succinyl-CoA
NADH
α-ketoglutaratedehydrogenase
complex
Oxidation of α-ketoglutarate to succinyl-CoA and CO2
CH2
CH2
COO-
C
O
S-CoA
CH2
CH2
COO-
COO-
ΔG'º = −2.9 kJ/mol
Succinyl-CoA
succinyl-CoAsynthetase
Succinate
CoA-SHGTPGDP + Pi
Conversion of succinyl-CoA to succinate
His
His His
CH2
CH2
C
CO O-
O S-CoA
CH2
CH2
COO-
COO-
CH2
CH2
C
CO O-
O O P
His P
Succinyl-CoA
succinyl-CoAsynthetase
Enzyme-boundsuccinyl
phosphate
Succinate
Phosphoenzyme
1 32
CoA-SH
Pi
GDP GTP
Succiny-CoA synthase reaction
CH2
CH2
COO-
COO-
CH
CH
COO-
COO-
ΔG'º = 0 kJ/mol
succinatedehydrogenase
Succinate Fumarate
FAD FADH2
Oxidation of succinate to fumarate
C
C
COO-
COO-
H
H CH
C
COO-
COO-
HO
H H
ΔG'º = -3.8 kJ/mol
fumarase
L-MalateFumarate
H2O
Hydration of fumarate to malate
NADH + H+
ΔG'º = 29.7 kJ/mol
malatedehydrogenase
CO
CH2
COO-
COO-
Oxaloacetate
NAD+
L-Malate
CH
C
COO-
COO-
HO
H H
Oxidation of malate to oxaloacetate
FADH2
NADH
GTP
CO2
NADH
NADH
Isocitrate
α-Ketoglutarate
Succinyl-CoA
Succinate
Fumarate
Malate
Oxaloacetate
Acetyl-CoA
Citrate
CO2
Products of one turn of the citric acid cycle
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 Glyceraldehyde 3-phosphate → 2 1,3-bisphosphoglycerate
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
Total 30-32
*This is calculated as 2.5 ATP per NADH and 1.5 ATP per FADH2. A negative value indicates consumption.
GlutamineProlineArginine
Phosphoenolpyruvate(PEP)
AspartateAsparagine
Porphyrins,heme
Fatty acids,sterols
pyruvate
pyruvate
Glutamate
Purines
malicenzyme
Pyrimidines
Glucose
SerineGlycineCysteine
PhenylalanineTyrosine
Tryptophan
pyruvatecarboxylase
PEP carboxylase
PEP carboxylase
Oxaloacetate Citrate
α-Ketoglutarate
Succinyl-CoA
Malate
Acetyl-CoA
Role of the citric acid cycle in anabolism
Anaplerotic Reactions
Reaction Tissue(s)/organism(s)
Liver, kidney
Heart, skeletal muscle
Plants, yeast, bacteria
Eukaryotes and prokaryotes
Phosphoenolpyruvate + CO2 + GDP oxaloacetate + GTPPEP carboxykinase
Pyruvate + HCO3- + ATP oxaloacetate + ADP + Pi
pyruvate carboxylase
Pyruvate + HCO3- + NAD(P)H Malate + NAD(P)+
malic enzyme
Phosphoenolpyruvate + HCO3- oxaloacetate + Pi
PEP carboxylase
N NH
S(CH2)3 CH2
OH
C
NH
O
Lys
N NH
S(CH2)3 CH2
OH
C
NH
O
Lys
Bicarbonate
enol form
Oxaloacetate
ATP
ADP
Carbondioxide
pyruvatecarboxylase
pyruvate
keto form
H+
Pi
+
Step 1
Step 2
C
O
OH-O
C
O
O
C
O
O HOP
O
O-
HO
N NH
S(CH2)3 CH2
O
C
NH
O
CO
-O
Lys
C
O
OHC C
-O
O O
CH2
C C-O
O O-
CH2
N NH
S(CH2)3 CH2
O
C
NH
O
CO
-O
LysC C
-O
O O
CH2-
C C-O
O O
C
H
H
H
The role of biotin in pyruvate carboxylase reaction
FAD
SH
SH
FAD
S
S
NAD+
NADH
4
5
Pyruvate
Hydroxyethyl-TPP
TPP
1
CO2
2
Acetyl-CoA
CoA
3
Product inhibition
Lipoamide
Dihydrolipoamide
Acetyl-dihydrolipoamide
E1 E2
E3
Covalent modification
H2O
Pi
pyruvatedehydrogenase
phosphatase
ATP
ADP
pyruvatedehydrogenasekinase
E1−OH (active)
E1−OPO32− (inactive)
Regulation of PDH complex
NADH
GTP
Isocitrate
Malate
Oxaloacetate
Citrate
FADH2
ATP
Succinyl-CoA
α-Ketoglutarate
Fumarate
Succinate
Pyruvate
Acetyl-CoA
malatedehydrogenase
pyruvatedehydrogenase
complex
citratesynthase
isocitratedehydrogenase
succinatedehydrogenase
ATP, acetyl-CoA,NADH, fatty acids
AMP, CoA, NAD+, Ca2+
ATP
ADP
NADH, succinyl-CoA, citrate, ATP
Ca2+
succinyl-CoA, NADH
Ca2+, ADP
α-ketoglutaratedehydrogenase
Regulation of metabolite flow through the citric acid cycle
1. Pyruvate, the product of glycolysis, is converted to acetyl-CoA, the starting material for the citric acid cycle, by the pyruvate dehydrogenase multienzymecomplex
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 acetyl-CoA 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, isocitratedehydrogenase, and α-ketoglutarate dehydrogenase