Citric Acid Cycle (VPP3021) PDF

Citric Acid Cycle Mokrish Ajat Dept. of Veterinary Preclinical Sciences Faculty of Veterinary Medicine UPM [email protected] VPP3021 Veterinary Biochemistry  What are w...

Citric Acid Cycle Mokrish Ajat Dept. of Veterinary Preclinical Sciences Faculty of Veterinary Medicine UPM [email protected] VPP3021 Veterinary Biochemistry  What are we going to discuss? Krebs cycle localization PDC (pyruvate dehydrogenase complex) Krebs cycle Regulation of Krebs cycle VPP3021 Veterinary Biochemistry  Overview and brief history Pyruvate Dehydrogenase Complex (PDC) and its control Reactions of TCA cycle/CAC/Krebs cycle Regulation of TCA cycle VPP3021 Veterinary Biochemistry  Reactions of Glycolysis are localized in Cytosol whereas pyruvate dehydrogenase and TCA cycle reactions take place in mitochondria where oxygen is utilized to generate ATP by oxydative phosphorylation. Consumption of oxygen (respiration) depends on the rate of PDC and TCA reactions. VPP3021 Veterinary Biochemistry  In Cytosol In Mitochondria VPP3021 Veterinary Biochemistry VPP3021 Veterinary Biochemistry  Historical perspective: 1930: Elucidation of Glycolysis Study of oxidation of glucose in muscle, addition of Malonate inhibited the respiration (i.e. O2 uptake). Malonate is an inhibitor of Succinate oxidation to Fumerate 1935: Szent-Gyorgyi: demonstrated that little amounts (catalytic amounts) of succinate, fumerate, malate or oxaloacetate acelerated the rate of respiration. He also showed the sequence of inter-conversion: Succinate --- Fumerate --- malate ---oxaloacetate. 1936: Martius & Knoop: Found the following sequence of reaction: Citrate to cis-aconitase to Isocitrate to a Ketogluterate to succinate 1937: Krebs: Enzymatic conversion of Pyruvate + Oxaloacetate to citrate and CO2 Discovered the cycle of these reactions and found it to be a major pathway for pyruvate oxidation in muscle. VPP3021 Veterinary Biochemistry VPP3021 Veterinary Biochemistry  Pyruvate dehydrogenase Complex (PDC) It is a multi-enzyme complex containing three enzymes associated together non- covalently: E-1 : Pyruvate dehydrogenase , uses Thiamine pyrophosphate as cofactor bound to E1 E-2 : Dihydrolipoyl transacetylase, Lipoic acid bound, CoA as substrate E-3 : Dihydrolipoyl Dehydrogenase FAD bound, NAD+ as substrate Advantages of multienzyme complex: 1. Higher rate of reaction: Because product of one enzyme acts as a substrate of other, and is available for the active site of next enzyme without much diffusion. 2. Minimum side reaction. 3. Coordinated control. VPP3021 Veterinary Biochemistry VPP3021 Veterinary Biochemistry  Reaction of pyruvate dehydrogenase complex (PDC) VPP3021 Veterinary Biochemistry  Please refer to text book page 794 and 795 (Voet), read it and expand this part of your notes. Five steps reaction involved in the formation of Acetyl-CoA. VPP3021 Veterinary Biochemistry VPP3021 Veterinary Biochemistry  Thiamin (Vitamine B1) deficiency in animals: Christiaan Eijkmann, Nobel Prize Winner 1929, Physiology and Medicine. Discovery of Vitamin B1 https://www.nobelprize.org/prizes/medicine/19 29/eijkman/biographical/ https://www.forbes.com/sites/kionasmith/2018/ 08/11/meet-the-dutch-army-doctor-who- discovered-vitamin-b-and-cured-a-deadly- disease/#4dbf00f15ef6 https://www.youtube.com/watch?v=X9qnaFTJ h5Y VPP3021 Veterinary Biochemistry  Thiamin (Vitamine B1) deficiency causes Beriberi: Thiamine pyrophosphate (TPP) is an important cofactor of pyruvate dehydrogenase complex, or PDC a critical enzyme in glucose metabolism. Thiamine is neither synthesized nor stored in good amounts by most vertebrates. It is required in the diets of most vertebrates. Thiamine deficiency ultimately causes a fatal disease called Beriberi characterized by neurological disturbances, paralysis, atrophy of limbs and cardiac failure. Note that brain exclusively uses aerobic glucose catabolism for energy and PDC is very critical for aerobic catabolism. Therefore thiamine deficiency causes severe neurological symptoms. VPP3021 Veterinary Biochemistry VPP3021 Veterinary Biochemistry  Reactions of TCA cycle: 8 reactions: 1. Citrate synthase 2. Aconitase 3. Iso-citrate dehydrogenase 4. a ketoglutarate dehydrogenase 5. Succinyl- Coenzyme A synthetase 6. Succinate dehydrogenase 7. Fumerase 8. Malate dehydrogenase http://higheredbcs.wiley.com/legacy/college/voet/0470570954/animated_figs/4e_ch21/21-25_4e.html VPP3021 Veterinary Biochemistry  Reactions of Citric Acid Cycle 1. Citrate synthase: Formation of Citroyl CoA intermediate. 2. Binding of Oxaloacetate to the enzyme results in conformational change which facilitates the binding of the next substrate, the acetyl Coenzyme A. There is a further conformational change which leads to formation of products. This mechanism of reaction is referred as induced fit model. VPP3021 Veterinary Biochemistry  2. Aconitase: This enzyme catalyses the isomerization reaction by removing and then adding back the water ( H and OH ) to cis-aconitate in at different positions. Isocitrate is consumed rapidly by the next step thus deriving the reaction in forward direction. VPP3021 Veterinary Biochemistry 3. Isocitrate dehydrogenase: There are two isoforms of this enzyme, one uses NAD+ and other uses NADP+ as electron acceptor. VPP3021 Veterinary Biochemistry  4. a-Ketoglutarate dehydrogenase: This is a complex of different enzymatic activities similar to the pyruvate dyhdogenase complex. It has the same mechanism of reaction with E1, E2 and E3 enzyme units. NAD+ is an electron acceptor. VPP3021 Veterinary Biochemistry  5. Succinyl CoA synthetase: Succinyl CoA, like Acetyl CoA has a thioester bond with very negative free energy of hydrolysis. In this reaction, the hydrolysis of the thioester bond leads to the formation of phosphoester bond with inorganic phosphate. This phosphate is transferred to Histidine residue of the enzyme and this high energy, unstable phosphate is finally transferred to GDP resulting in the generation of GTP. VPP3021 Veterinary Biochemistry 6. Succinate Dehydrogenase: Oxidation of succinate to fumarate. This is the only citric acid cycle enzyme that is tightly bound to the inner mitochondrial membrane. It is an FAD dependent enzyme. Malonate has similar structure to Succinate, and it competitively inhibits SDH. VPP3021 Veterinary Biochemistry 7. Fumarase: Hydration of Fumarate to malate: It is a highly stereospecific enzyme. Cis-Maleate (the cis form of fumarate is not recognized by this enzyme. VPP3021 Veterinary Biochemistry 8. L-Malate dehydrogenase: Oxidation of malate to oxaloacetate: It is an NAD+dependent enzyme. Reaction is pulled in forward direction by the next reaction (citrate synthase reaction) as the oxaloacetate is depleted at a very fast rate. VPP3021 Veterinary Biochemistry Regulation of CAC: Rate controlling enzymes: Citrate synthatase Isocitrate dehydrogenase a-ketoglutaratedehydrogenase Regulation of activity by: Substrate availability Product inhibition Allosteric inhibition or activation by other intermediates http://higheredbcs.wiley.com/legacy/college/v oet/0470570954/animated_figs/4e_ch21/21- 1_4e.html VPP3021 Veterinary Biochemistry Pathways that utilize TCA intermediates 1. Glucose biosynthesis (gluconeogenesis) 2. Lipid biosynthesis 3. Amino acid biosynthesis 4. Porphyrin biosynthesis 5. Complete oxidation of amino acids http://higheredbcs.wiley.com/legacy/college/v oet/0470570954/animated_figs/4e_ch21/21- 26_4e.html VPP3021 Veterinary Biochemistry  Watch videos on my youtube channel, playlist Krebs Cycle Browse these links for guided explorations and exercises http://higheredbcs.wiley.com/legacy/college/voet/0470570954/guided_exps/4e_ch21_g ex_18/citric_acid_cycle_4e.html VPP3021 Veterinary Biochemistry  What have we discussed? Krebs cycle localization PDC Krebs cycle Regulation of Krebs cycle VPP3021 Veterinary Biochemistry