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