Kreb's cycle

1. PYRUVATE OXIDATION & KREB’S CYCLE PRESENTED BY: NOOPUR JOSHI M.Sc. Biotechnology

2. CELLULAR RESPIRATION Under aerobic conditions, the cells obtain energy from ATP, produced as a result of breakdown of glucose. The aerobic organisms oxidize their organic fuels completely to CO₂ and H₂O. In such conditions, the pyruvate, instead of being reduced to lactate, ethanol and CO ₂, gets completely oxidized in to CO₂ and H₂O. This is termed as Cellular respiration.

3. Thus Cellular respiration can be defined as: “ A sequence of molecular processes involved in O₂ consumption and CO₂ formation by the cells.”

4. 3 STAGES OF CELLULAR RESPIRATION STAGE 1: “Oxidative decarboxylation of Pyruvate to Acetyl CoA and CO₂.” This conversion is catalyzed by a highly organized multienzyme “ pyruvate dehydrogenase complex.” In the overall reaction, the carbooxylic group of pyruvate is lost as CO ₂, while the remaining 2 carbons form the acetyl moeity of acetyl- CoA . The reaction is highly Exergonic and is essentially irreversible, in vivo.

5. CO ₂ NAD⁺ NADH

6. STAGE 2: “Citric acid Cycle or Acetyl CoA catabolism” In this stage, the acetyl group so obtained is fed into citric acid cycle/ Kreb’s Cycle which then degrades it to yield energy rich hydrogen atoms and to release CO₂; the final product of organic fuels. It is the final common pathway for oxidation of fuel molecules. This cycle also provides intermediates for biosynthesis.

7. STAGE 3: “Electron transport chain and oxidative phosphorylation” In this final stage of respiration, the hydrogen atoms are separated into protons (H⁺) and energy rich electrons. The electrons are transferred via chain of electron-carrying molecules, the respiratory chain, to molecular oxygen, which is reduced by electrons to form water.

8. PYRUVATE OXIDATION The oxidative decarboxylation of pyruvate to form Acetyl CoA Is the link between glycolysis and kreb’s cycle. It occurs in mitochondrial matrix. Here pyruvate from Glycolysis is dehydrogenated to form Acetyl CoA and CO₂ by the enzyme pyruvate dehydrogenase complex. The reaction is irreversible and can be represented as follows:

9. COO ‾ S CoA C O + CoA SH + NAD⁺ C O + CO₂ + NADH CH₃ CH₃ Pyruvate dehydrogenase complex Mg²⁺ Pyruvate Acetyl CoA Coenzyme A

10. This conversion is catalyzed by a highly organized multienzyme “ pyruvate dehydrogenase complex.” In the overall reaction, the carbooxylic group of pyruvate is lost as CO ₂, while the remaining 2 carbons form the acetyl moeity of acetyl-CoA. The reaction is highly Exergonic and is essentially irreversible, in vivo.

11. KREB’S CYCLE Also known as Citric acid cycle was discovered by H.A.Kreb , German born British Biochemist. This cycle occurs in mitochondrial matrix in eukaryotes and in cytosol in prokaryotes. The net result for this cycle is that for each acetyl group entering the cycle as Acetyl CoA, 2 molecules of CO₂ are produced.

12. Acetyl CoA CoA-SH Aconitase + H₂O Aconitase + H₂O Isocitric Dehydrogenase + Mn²⁺ CO₂ NADH+H⁺ NAD⁺ Succinyl CoA Synthetase + Mg²⁺ Succinic dehydrogenase NADP⁺ NADPH+ H⁺ Fumerase Malic dehydrogenase NADH+ H⁺ NAD⁺ FADH₂ FAD +CO₂ H₂O

13. STEP WISE EXPLAINATION OF THE CITRIC ACID/TRICARBOXYLIC/KREB’S CYCLE

14. STEP1 : Condensation OF Acetyl-CoA with Oxaloacetate The cycle begins with the condensation of a 4 carbon unit, the oxaloacetate , and the acetyl group of the Acetyl CoA, which is a 2 carbon unit. Oxaloacetate reacts with Acetyl-CoA and H₂O to yield citrate and CoA. This reaction is an aldol condensation reaction and is followed by hydrolysis.itis catalyzed by the enzyme: “ citrate synthetase”.

15. CITRATE SYNTHETASE H₂O CONDENSATION HYDROLYSIS Citryl -CoA Oxaloacetate Acetyl-CoA Citrate Coenzyme-A H + + +

16. STEP 2: ISOMERIZATION OF Citrate INTO Iso -citrate In this reaction, water is first removed and then added back, moves the hydroxyl group from one carbon atom to its neighbor. The enzyme catalyzing this reaction is aconitase. CITRATE Aconitase -H₂O Cis - ACONITATE +H₂O ISOCITRATE Aconitase -H₂O +H₂O

17. STEP 3: Oxidative Decarboxylation of Isocitrate Isocitrate is oxidized and decarboxylated into α - ketogluterate . This reaction is catalyzed by the enzyme “ isocitrate dehydrogenase.” ISOCITRATE OXALO-SUCCINATE (enzyme bound) α - KETOGLUTERATE NAD⁺ NADH+H⁺ H⁺ CO₂ Isocitrate dehydogenase Isocitrate dehydogenase

18. STEP 4: Oxidative decarboxylation of α - ketogluterate This second oxidative decarboxylation results in formation of “ Succinyl CoA” from α - ketogluterate . “ α - ketogluterate dehydrogenase” catalyzes this oxidative step and produces NADH, CO₂ and a high-energy thioester bond to coenzyme-A (CoA).

19. α - ketogluterate + CoA—SH NAD⁺ + α - ketogluterate Dehydrogenase complex Succinyl -CoA

20. STEP 5: Conversion of Succinyl -CoA into Succinate The cleavage of the thioester bond of Succinyl -CoA is coupled to the phosphorylation of a purine nucleoside diphosphate , usually GDP ( substtrate level phosphorylation). It is catalyzed by “ succinyl CoA synthetase/ succinyl thiokinase ”. This is the only step in the Kreb’s Cycle that directly yields a compound with high phosphoryk transfer potential through a substrate level phosphorylation

21. Succinyl -CoA Succinyl phosphate (enzyme bound) Succinate Pi CoA—SH Mg²⁺ Mg²⁺

22. STEP 6: Dehydrogenation of Succinate to form Fumerate In this third oxidation step, FAD removes 2 hydrogen atoms from succinate. This reaction is catalyzed by the enzyme “succinate dehydrogenase.” This reaction is the only dehydrogenation in the citric acid cycle in which NAD⁺ doesn’t participate. Rather, hydrogen is directly transferred from the substrate to falvoprotein enzyme (succinate dehydrogenase).

23. Succinate Fumerate E—FAD + Succinate dehydrogenase

24. STEP 7: Hydration of Fumerate to Malate Fumerate is hydrated to form L- malate in the presence of “ fumerate hydratase ”. It involves hydration i.e. addition of water to fumerate which places a hydroxyl group next to the carbonyl carbon.

25. Fumerate L- malate Fumerate hydratase +H₂O -H₂O

26. STEP 8: Dehydrogenation of Malate to Oxaloacetate This is the 4 th oxidation-reduction reaction in the citric acid cycle where L- malate is dehydrogenated to oxaloacetate . This reaction takes place in the presence of “l L - malatae dehydrogenase”. The NAD⁺ which remains linked to the enzyme molecule acts as the hydrogen acceptor and gets reduced to NADH and H⁺ This reaction is a reversible reaction.

27. Although the equilibrium of this reaction favours formation of malate again, but the reaction proceeds forward since the oxaloacetate and the NADH so formed are removed rapidly and continuously in the further reactions. The generated Oxaloacetate allows repetition of the cycle and NADH precipitates in oxidative phosphorylation This reaction completes the cycle.

28. COO‾ HO C H H C H COO ‾ L- Malate + NAD⁺ COO ‾ C O CH₂ COO ‾ + + NADH H⁺ OXALOACETATE L- malate dehydrogenase

29. Thus, the complete cycle so obtained can be represented as follows:

30. Acetyl CoA CoA-SH Aconitase + H₂O Aconitase + H₂O Isocitric Dehydrogenase + Mn²⁺ CO₂ NADH+H⁺ NAD⁺ Succinyl CoA Synthetase + Mg²⁺ Succinic dehydrogenase NADP⁺ NADPH+ H⁺ Fumerase Malic dehydrogenase NADH+ H⁺ NAD⁺ FADH₂ FAD +CO₂ H₂O

31. THANK YOU