The citric acid cycle : Thecatabolism of Acetyl-CoA 17

• Citric acid cycle에서 조절에 관여하는 반응 단계와 조절기전을 설명한다.

• 글루코스 한 분자가 세포내에서 완전히 산화되었을 때 생성되는 ATP 갯수를 각 과정별로

산출하여 설명한다.

• 탄수화물, 단백질, 지방의 대사 경로와 상호작용을 설명한다.

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Outline of the pathways for the catabolism of dietary carbohydrate, protein, and fat2

3

4Overview of glycolysis pathway

D-glucose

Pyruvate`

pyruvate

Acetyl-CoA

In aerobic condition, glycolysis is terminated at pyruvate

5

glucose 6-ⓟ

pyruvate

glucose

glycogen

PEP

oxaloacetate

acetyl-CoA

oxaloacetate

fatty acid

TG

pyruvate acetyl-CoA

1

2

3

mitochondriacytosol

• Acetyl-CoA는 mito. memb에 impermeable.• Aconitase가 saturation된경우 citrate는 mito.밖으로나올수있다.

(1) pyruvate symporter(2) malate shuttle(3) carnitine transporter

BIOMEDICAL IMPORTANCE

Citric acid cycle (Krebs cycle, TCA cycle)

• Reactions in mitochondria

• Acetyl residue oxidizes to CO2 and produces high energy products

• Final common pathway of CHO, lipid and protein

• Central role in gluconeogenesis, lipogenesis, interconversion of AAs

• The liver is the only tissue in which all occur to a significant extent

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THE CIRIC ACID CYCLE PROVIDES SUBSTRATE FOR THE RESPIRATORY CHAIN

• Acetyl-CoA + 3NAD+ + FAD + ADP + Pi + 2H2O

→ CoA-SH + 2CO2 + 3NADH + 3H+ + FADH2 + ATP

• Respiratory chain

: oxidation of coenzymes coupled with

formation of ATP

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Citric acid cycle itself :

no consume oxygen.

produce very little ATP

Citr

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cycl

e ov

ervi

ew

Figure 17-3

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*

*

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*

*

*

*

*

*

*

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*

*

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REACTIONS OF THE CIRIC ACID CYCLE LIBERATE REDUCING EQUIVALENTS & CO2

Acetyl-CoA (C2) + Oxaloacetate (C4) → Citrate(C6) → Oxaloacetate (C4) + 2CO2

: Oxidized coenzymes → Reduced coenzymes

• Acetyl-CoA + 3NAD+ + FAD + ADP + Pi + 2H2O

→ CoA-SH + 2CO2 + 3NADH + 3H+ + FADH2 + ATP (or GTP)

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Stero-selectivity - asymmetric reaction in symmetric substrate

- It result from the serial reaction between closely related enzymes (channeling)

Citrate synthase• Substrate : acetyl-CoA (C2) and oxaloacetate (C4) / Product : citrate (C6)

• Release CoA-SH & hydration reaction, irreversible (ΔG= -7.5 kcal/mol)

• Citrate synthase is produced in cytoplasm and transported to the mitochondria.

Pyruvate (C3)

PDH

pyruvatecarboxylase

****

******

**

* *

****

CoA

+CO2

+H2O

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Aconitase• Substrate : citrate / Product : aconitate, isocitrate

• Dehydration and Rehydration reactions

• Despite of symmetrical shape of citrate, asymmetrical reaction is the result of channeling

- transfer directly the citrate from citrate synthase to aconitase

- subsequent reactions leave the carbons derived from acetyl-CoA

• nuclear gene encoding mitochondrial protein

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Aconitase Inhibitor

※ Sodium fluoroacetate and fluorocitrate are toxic to herbivores and 2’ carnivores

• fluoroacetate fluoroacetyl-CoA fluorocitrate

- fluorocitrate bind to aconitase tightly

- decreased aconitase activity

- citrate accumulation

CoA oxaloacetate

citrate synthase

poison plant containing sodium fluoroacetate12

1080

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포섬

Isocitrate dehydrogenase (IDH)• Substrate : isocitrate / Product : α-KG

• Two functional enzyme : Dehydrogenation and Decarboxylation

• 3 isozymes

- IDH 1 & 2 : NADP+-dependent, mitochondria & cytosol

- IDH 3 : NAD + -dependent, mitochondria

• nuclear gene encoding mitochondrial protein

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Isocitrate dehydrogenase (IDH)• Specific mutations are found in brain tumors

: astrocytoma, oligodendroglioma and glioblastoma multiforme

: impaired citric acid cycle & glycolysis dependent tumor

• The relation is not clear between the IDH1 mutation and development of glioblastoma multiforme.

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α-ketoglutarate dehydrogenase complex

• Substrate : α-KG / Product : succinyl-CoA

• Similar PDH, irreversible (ΔG= -7.2 kcal/mol)

• A-KGDH complex is a key control point in the citric acid cycle.

• Cofactors : Coenzyme A, thiamin diphosphate, FAD, lipoate, NAD +

• Inhibition by succinyl-CoA and NADH

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α-ketoglutarate dehydrogenase complex

• Toxicity of arsenite (As2O3)As

As

O O O

Ez Ez

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- React with -SH group of enzyme and block the catalytic site

Succinate thiokinase (succinyl-CoA synthetase)

• Substrate : succinyl-CoA / Product : succinate

• Reversible, ATP or GTP production (GTP readily convert to ATP)

• isozymes

- GDP dependent : liver & kidney

- ADP dependent : all tissues

※ PEP carboxykinase in gluconeogenesis (cytosol) use this GTP

- oxaloacetate + GTP → phosphoenolpyruvate + GDP

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Succinate thiokinase (succinyl-CoA synthetase)

• Succinyl-CoA is necessary for synthesis of porphyrin, heme, and ketone bodies.

* thiol group : organo-sulfurhydryl compound (R-SH)

• CoA transferase (thiophorase) in β-oxidation (Fig. 22-8): Liver

FFA →→ acetyl-CoA ↔ HMG-CoA ↔ acetoacetate (ketone body)

: Extrahepatic tissues

acetoacetate + succinyl-CoA ↔ acetoacetyl-CoA + succinate → acetyl-CoA

• Acetoacetate is released from liver, acetoacetate is cleaved to acetyl-CoA by

succinyl-CoA-acetoacetate CoA transferase. → Ketone bodies serve as a fuel for

extrahepatic tissues

• Fatal infantile lactic acidosis due to defect succinyl-CoA synthetase.

*

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Succinate dehydrogenase• Substrate : succinate / Product : fumarate

• Succinate dehydrogenase, Succinate Q reductase, complex II

• Coenzyme

- FAD, (Fe:S) protein directly reduce ubiquinone (UQ, coenzyme-Q) and transfer the electron

to respiratory chain.

- produce 1.5 ATP per 1 FADH2 (P:O=1.5)

*

Inner mitochondrialmembrane

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4 4 2

Succinate dehydrogenase inhibitor• malonate

- a competitive inhibitor

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Fumarase• Substrate : fumarate / Product : malate

• Hydration reaction

• Present in both of mitochondria and cytosol.

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Malate dehydrogenase

• Substrate : malate / Product : oxaloacetate

• Coenzyme : NAD+

• The equilibrium of this reaction strongly favors malate (ΔG= 7.1 kcal/mol)

• Consumption of oxaloacetate : (oxaloacetate concentration is very low)

: oxaloacetate → → pyruvate in gluconeogenesis

: oxaloacetate → aspartate in transaminase

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Malate dehydrogenase

• Malate shuttle (Fig. 13-13)

- transfer NADH (through inner mitochondrial membrane)

- impermeability of NADH

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Malate dehydrogenase

• Glycerophosphate shuttle (Fig. 13-12)

- GP shuttles consume NADH in cytosol and generate FADH2 in mitochondria

- When use GP shuttle, P:O ratio of NADH is 1.5

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TEN ATP ARE FROMED PER TURN OF THE CIRIC ACID CYCLE

Acetyl-CoA (C2) + Oxaloacetate (C4) → Citrate(C6) → Oxaloacetate (C4) + 2CO2

: Oxidized coenzymes → Reduced coenzymes

• Acetyl-CoA + 3NAD+ + FAD + ADP + Pi + 2H2O

→ CoA-SH + 2CO2 + 3NADH + 3H+ + FADH2 + ATP

NADH : ~2.5 ATP

FADH2 : ~1.5 ATP

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• Full oxidation of glucose

: glycolysis + pyruvate oxidation + citric acid cycle + respiratory chain

• Glucose + 2 H2O → 6 CO2 + 6 H+ + 32ATP

Citric acid cycle and respiratory chain

pathway enzymes Method of ATP formation ATP per 1 glucose molecule

glycolysisGlyceraldehyde 3-phosphate dehydrogenase

Respiratory chain oxidation of 2 NADH 5*

Phosphoglycerate kinase Substrate level phosphorylation 2

Pyruvate kinase Substrate level phosphorylation 2

Hexokinase/PFK Consumption of ATP -2

Net 7

Citric acid cycle

Pyruvate dehydrogenase Respiratory chain oxidation of 2 NADH 5

Isocitrate dehydrogenase Respiratory chain oxidation of 2 NADH 5

A-KG dehydrogenase Respiratory chain oxidation of 2 NADH 5

Succinate thiokinase Substrate level phosphorylation 2

Succinate dehydrogenase Respiratory chain oxidation of 2 FADH2 3

Malate dehydrogenase Respiratory chain oxidation of 2 NADH 5

Net 25Fig. 18-1 * NADH transfer to mitochondria by the malate shuttle. The glycerophosphate shuttle formed 1.5 ATP per NADH 27

VITAMINS PLAY KEY ROLES INTHE CIRIC ACID CYCLE

• 4 B vitamins

(1) Riboflavin (to form of FAD, vit. B2)

(2) Niacin (to from of NAD, vit. B3)

(3) Thiamin (vit. B1; to form of TDP)

(4) Pantothenic acid (vit. B5; a part of CoA)

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THE CIRIC ACID CYCLE PLAYS A PIVOTAL ROLEIN METABOLISM

• Oxidation of acetyl-CoA

• Amino acids synthesis : transamination / deamination

• Amino acids degradation : gluconeogenesis

• Fatty acid synthesis : lipogenesis

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Gluconeogenesis

• chapter 20

• Pyruvate carboxylase

- substrate : pyruvate / product : oxaloacetate

- biotin cofactor

- pyruvate ▷ pyruvate dehydrogenase ▷ acetyl-CoA

▶ pyruvate carboxylase ▶ oxaloacetate

pyruvate (C3) oxaloacetate (C4)CO2 ATP ADP + Pi

Pyruvate carboxylase

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Gluconeogenesis

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MALATE DEHYDROGENASE

• Malate dehydrogenase

• Phospoenolpyruvate carboxykinase

- substrate : oxaloacetate / product : PEP

- GTP consumption (prevent excessive consumption of oxaloacetate)

asparagine

Transaminase• Form pyruvate from alanine, oxaloacetate, aspartate, a-KG

• Reversible : amino acid degradation & synthesis

PDH

LeucinePhenylalanineTryptophan

tyrosinelysine

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pyruvateglucose

Acetoacetyl-CoA

IsoleucineLeucine

Tryptophan

HGAP

I,m V

Transaminase

AST

ALT

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Propionate• Propionate is major glucose source in ruminants

• Propionate arise from odd-chain fatty acids in ruminant lipids

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Fatty acid synthesis• Long chain fatty acid synthesized from acetyl-CoA

• Acetyl-CoA is impermeable mitochondrial membrane

• Citrate is available for transport out of the mitochondrial membrane when aconitase is

saturated with its substrate.

• Citrate is cleaved to oxaloacetate and acetyl-CoA

• acetyl-CoA is used for fatty acid in cytosol

aconitase

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Regulation of the citric acid cycle• Activity of citric acid cycle is dependent on the supply of NAD+ and the availability of

ADP

• The nonequilibrium reactions : (PDH), citrate synthase, isocitrate dehydrogenase, α-

KG dehydrogenase

• PDH◁ [ATP]/[ADP], [NADH]/[NAD+]

• Citrate synthase◁ATP, acyl-CoA

• Isocitrate dehydrogenase◀ADP◁ATP, NADH

• α-KG dehydrogenase◁ [ATP]/[ADP], [NADH]/[NAD+]

• Succinate dehydrogenase◁ oxaloacetate

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CLINICAL ASPECT• Hyperammonemia due to liver diseases and defect of amino acid metabolism

: enhanced conversion α-KG to glutamate

→ reduced intermediates of all citric acid cycle

→ reduced generation of ATP → cell swelling

: loss of consciousness and convulsions (hepatic coma, hepatic encephalopathy)

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1. Acetyl-CoA, common end-metabolite of carbohydrate,

lipid and protein

2. Reduced coenzymes is oxidized

- formation of ATP

3. Citric acid cycle is amphibolic

- gluconeogenesis, fatty acid synthesis

- interconversion of amino acids

Department of Biochemistry Pusan National University School of Medicine