Post on 20-Oct-2015
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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
ic a
cid
cycl
e ov
ervi
ew
Figure 17-3
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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
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
17
- 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