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- 1. Oxidative Decarboxylation of Pyruvate & Krebs Cycle Dr. Endriyas Kelta (DMD, MSc) Assistant Professor, AAU 1
- 2. Citric Acid Cycle – Also known as: • Tricarboxylic acid (TCA ) cycle –After its substrates, Citrate & Iso-citrate • Krebs cycle –(After its discoverer, Hans Krebs) – Special metabolic pathway which runs in mitochondria • Most of our cells contain mitochondria which act as “mobile power plant” • Few cells however, don’t have mitochondria –E.g., Red Blood cells (do not run Krebs’ cycle ) • All the enzymes which mediate the cycle present within mitochondria 2
- 3. Cont… Mitochondria: – Intracellular organelles which are bounded by double membranes • Outer membrane • Inner membrane – What was origin of mitochondria? • Originally, mitochondria were energy producing bacteria • Millions’ of years back, visited eukaryotic cell – Eukaryotic cell provided protection & nutrition to mitochondria – Bacteria were very happy & provided energy – What is proof that mitochondria were bacteria? • Like bacterial DNA, mitochondrial DNA is circular 3
- 4. Cont… – Inter-membraneous space – Matrix • All the substances in the inner side of inner membrane – What are substances present in the mitochondrial matrix? » Mitochondrial DNA » Mitochondrial RNA » Mitochondrial Ribosomes » Krebs cycle enzyme » Pyruvate dehaydrogenase complex » FA beta oxidation enzymes 4
- 5. Cont… – What are substances present in the mitochondrial inner membrane? » ETCS protein complex » Mobile electron carriers: Co-Q & cytochrome C » Inner membrane particle responsible for ATP synthesis » Carrier proteins 5
- 6. Cont… – Cyclic process • Final product of pathway is exactly same to initial substrate What is the purpose of Krebs Cycle? – Concerned with: • Breakdown of acetyl moiety of Acetyl-CoA • Provision of intermediates to biosynthetic pathways • Catabolism of acetyl-CoA: – Acetyl-CoA is a major fuel for energy producing cycle (Krebs cycle) – Passes through cycle & catabolized, while releasing energy – From where Acetyl-CoA comes? – Breakdown product of carbon skeletons of major fuels in our body (Carbohydrates, Lipids & Proteins) 6
- 7. Sources of Acetyl-CoA in TCA cycle 7
- 8. Cont… • What happens when acetyl-CoA moves through the cycle? – Two carbon-units (acetyl moieties) of acetyl-CoA are lost as 2 molecules CO2 – Energy released is captured in the form of 3 molecules of NADH + H+ & one molecule of FADH2 – NADH + H+ & FADH2 molecules enter into electron transport chain system (ETCS) to regenerate NAD+ & FAD • Purpose of NADH + H+ & FADH2 molecules is to take energy rich electrons and protons from Krebs cycle to ETCS 8
- 9. Cont… • What will happen when energy rich electrons & protons are passing through ETCS? – Oxygen will be added to pathway at end, meets proton & H2O is produced – Energy released will be utilized to convert ADP + Pi into ATP • In summary, Krebs cycle: – Final common pathway of catabolism of major body feuls – Involves a sequence of dehydrogenation & de- carboxylation reactions • De-carboxylation helps blow out CO2 • Dehydrogenation helps transfer protons & electrons to NAD+ & FAD – Responsible to produce H2O & to oxidative phosphorylation of ADP into ATP 9
- 10. Reactions of Citric Acid Cycle – Junction Reaction: • Where is the cellular location of Krebs’ cycle? => Mitochondrion • Where is the cellular location of glcolysis? => Cytosol • How pyruvate can be transported from the cytosol into the mitochondrial matrix? => Pyruvate TOM & TIM • How pyruvate can be converted into Acetyl-CoA? => Oxidative decarboxylation • Junction reaction between Glcolysis and Krebs’ cycle 10
- 11. Overall reaction Pyruvate Oxidation 11
- 12. Oxidative Decarboxylation of Pyruvate12
- 13. Regulation of Junction reaction 13
- 14. Clinical Comments related to junction reaction • Congenital Lactic Acidosis: – X- linked dominant defect in PDH complex enzyme system – Can be of three types: • Severe – Neonatal (intra-uterine) death • Moderate – Severe mental retardation – Death in infancy • Milder – Persons may survive a little more – CNS dysfunction during carbohydrate meal » Failure in coordination of body movement => Episodic Ataxia – Mgt: Ketogenic diet 14
- 15. Cont… • Arsenic Poison: – Arsenate binds with Lipoic acid component of E2 – Produce very mild inhibition • Progressive accumulation of lactic acidDamage to CNS – Mgt: Dimercaprol which binds preferentially to arsenate than lipoic acid & excreted 15
- 16. Cont… • Citric Acid Cycle Reactions: – Condensation Reaction (Step-1) – Dehydration-rehydration Reactions (Step-2) – Oxidative Decarboxylation Reactions (Step-3 & Step-4) – Substrate Level Phosphorylation Reaction (Step- 5) – Dehydrogenation Reactions (Step-6 & Step-8) – Hydration Reaction (Step-7) 16
- 17. Reactions of Citric Acid Cycle 17
- 18. Step-1: Formation of Citric Acid (Condensation Reaction) – Oxaloacetate (4-C) condenses with acetyl CoA (2-C) – Citrate (6-C compound) is formed – Catalyzed by citrate synthase – Thioester bond in acetyl CoA is hydrolyzed – Hydrolysis of thioster bond drives the reaction forward – Irreversible reaction step 18
- 19. Cont… Step-2: Formation of Isocitrate (Dehydration / Rehydration Reaction) – Reversible reaction step – Citrate is isomerized to isocitrate – Catalyzed by aconitase (Inhibited by Fluoroacetate) – Two-step process: • Dehydration of citrate forming cis-aconitate • Rehydration of aconitate to form isocitrate 19
- 20. Cont… Step-3: Formation of Alpha Keto Glutarate (Oxidative Decarboxylation Reaction) – Irreversible reaction step – Isocitrate is oxidatively decarboxylated to α- keto glutarate – Catalyzed by isocitrate dehydrogenase – Two-step process: • Dehydrogenation of isocitrate forming oxalosuccinate • Decarboxylation of oxalosuccinate forming alpha keto glutarate – One molecule NADH + H+ is generated – One molecule of CO2 is liberated 20
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- 22. Cont… Step-4: Formation of Succinyl CoA (Oxidative Decarboxylation Reaction) – Irreversible reaction Step – Alpha keto glutarate is oxi-datively decarboxylated to succinyl CoA – Catalyzed by alpha keto glutarate dehydrogenase Complex • One molecule NADH + H+ is generated • One molecule of CO2 is liberated – Similar reaction mechanism to PDH complex 22
- 23. Cont… • Like PDH complex, alpha keto glutarate dehydrogenase complex – Contains 3 enzyme proteins and 5 co-enzymes • 3-enzme proteins: – Alpha keto glutarate dehydrogenase – Trans-Succinylase – Dihydrolipoyl dehydrogenase • 5 co-enzymes: – TPP, Lipoate, Co-enzyme A, FAD & NAD+ 23
- 24. PDH complex Vs α-KG-DH complex reactions 24
- 25. Cont… Step-5: Generation of Succinate (Substrate Level Phosphorylation Reaction) – Irreversible reaction step – Thioester bond of succinyl CoA is broken to form Succinate & high energy phosphate (GTP) – Catalyzed by succinate thiokinase (succinyl CoA synthetase) • One molecule of GDP is phosphorylated to GTP • GTP can be converted to ATP by reacting with an ADP molecule: – GTP + ADP → GDP + ATP (catalyzed by Nucleoside diphosphokinase) – What is difference b/n synthase & synthetase? 25
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- 27. Cont… Step-6: Formation of Fumarate (Dehydrogenation Reaction) – Reversible reaction step – Succinate is dehydrogenated to fumarate (unsaturated dicarboxylic acid) – Catalyzed by a flavoprotein enzyme, succinate dehydrogenase • One molecule of FADH2 is generated – Succinate dehydrogenase • Located in the inner side of inner mitochondrial membrane • Competitively inhibited by malonate 27
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- 29. Cont… Step-7: Formation of Malate (Hydration reaction) – Reversible reaction step – Hydration of fumarate to L-malate – Catalyzed by Fumarase • Specific to trans-double bond of fumarate & L-isomer of malate 29
- 30. Cont… Step-8: Regeneration of Oxaloacetate (Dehydrogenation Reaction) – Reversible reaction step – Malate is oxidized to oxaloacetate (may be viewed as a catalyst) – Catalyzed by malate dehydrogenase • One molecule of NADH + H+ is generated 30
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- 32. Overall energy balance of Krebs Cycle • By the help of Krebs cycle: – Large amount of energy is generated in the form of ATP via Oxidative phosphorylation • Accounts for 2/3rd of ATP generated from fuel oxidation – Small amount of energy is generated directly when GTP is converted into ATP via Substrate Level phosphorylation • ATP & GTP are Cash energy • NADH & FADH2 are Check Energy – Worth of one NADH is 3 ATP & FADH2 is 2 ATP 32
- 33. Cont… • Over all reaction equation for one round Krebs cycle – Acetyl-CoA+ 2H2O + 3NAD+ + FAD + GDP + Pi 2CO2 + GTP + 3NADH + 3H+ + FADH2 + CoASH – Fate of the acetyl moiety of Acetyl-CoA • One molecule of Acetyl-CoA Two molecules of CO2 – Fate of phosphoryl groups • One molecules of GDP & Pi one molecules of ATP – Fate of Electrons • Four pairs of electrons leave cycle in form of 3NADH & FADH2 • 12 ATP molecules are formed in one round of Krebs cycle after re-oxidation of 3NADH & FADH2 via ETCS 33
- 34. Cont… – Q1: When one molecule of glucose is gone into a cell, how much ATP will be produced in the presence of Oxygen? • Answer:36/38 ATP (How?) – Hint: • Glycolysis pathway = 6/8 ATP (why?) • Junction pathway= 6 ATP • Krebs’ cycle = 2 ATP via substrate level phosphorylation & 22 ATP via oxidative phosphorylation => 24 ATP 34
- 35. Glycerol 3-Phosphate Shuttle system (GPS) 35
- 36. Malate–Aspartate Shuttle system (MAS) 36
- 37. Regulation of Citric Acid Cycle (1)Respiratory control via ETCS & oxidative phosphorylation – Krebs Cycle runs only in presence of Oxygen => aerobic pathway – Respiratory control exerts main control on activities of Krebs cycle • E.g., Absence of O2 (anoxia) or partial deficiency of O2 (hypoxia) causes total or partial inhibition of cycle 37
- 38. Cont… (2) Regulation according to energy requirement of cell – E.g., During muscular contraction • Rise in intracellular Ca2+ level: – Stimulate multiple enzymes of cycle: » PDH complex (Junction reaction) » Citrate synthase » Isocitrate dehydrogenase » α-Ketoglutarate dehydrogenase • More ADP,AMP/ATP ratio – Stimulate multiple enzymes of cycle • More NAD+/NADH & FAD/FADH2 ratios – Stimulate multiple enzymes of cycle 38
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- 40. Link b/n TCA cycle & other metabolic pathways – TCA cycle termed as amphibolic pathway in nature • Has dual role – Catabolic » Concerned with degradation of Acetyl-CoA to generate ATP – Anabolic » Concerned with provision of intermediates for biosynthetic pathways • Biosynthesis of glucose • Fatty acid synthesis • Inter-conversion of Amino acids • Cholesterol & steroid synthesis 40
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- 42. Cont… • Anaplerotic Reactions – Reactions that replenish intermediates of TCA cycle • As TCA cycle intermediates efflux for biosynthetic pathways – 4 or 5-carbons compounds removed from cycle Depletes OAA levels Impossible to continue oxidizing acetyl-CoA Stoppage of krebs cycle • Cells supply enough 4 or 5-carbon intermediates – Degradation of carbohydrate – Degradation certain amino acids – Degradation of odd chain FAs 42
- 43. Major anaplerotic pathways of TCA cycle 43
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- 45. Cont… – Role of Vitamins in TCA Cycle • Five vitamin B-complexes are associated with TCA cycle: – Riboflavin used in: » FAD form • Coenzyme for Succinate DH – Niacin used in: » NAD+ form • Coenzyme for ICDH, α-KGDH & MDH – Thiamine used in: » TPP form • Coenzyme for α-KGDH – Lipoic acid used in: » Oxidized lipoamide (LS-SL) form & dihydrolipoamide (2-LSH) form • Coenzyme for α-KGDH – Pantothenic acid: Used as part of coenzyme A » Coenzyme for α-KGDH & Succinate thiokinase 45
- 46. Summary of Krebs Cycle 46
- 47. Thank you! 47