Lecture 2: Oxidative Decarboxylation and Krebs Cycle PDF

Oxidative decarboxylation and Krebs cycle Dr. Yasser Elghobashy Medical Biochemistry and Molecular Biology  Oxidative decarboxylation of pyruvate Pyruvate is an α-keto acid produced from glucose by glycolysis inside cytoplasm. To undergo oxidative decarboxylation into acetyl...

Oxidative decarboxylation and Krebs cycle Dr. Yasser Elghobashy Medical Biochemistry and Molecular Biology  Oxidative decarboxylation of pyruvate Pyruvate is an α-keto acid produced from glucose by glycolysis inside cytoplasm. To undergo oxidative decarboxylation into acetyl CoA it must be transported into the mitochondria by special pyruvate transporter. Oxidative decarboxylation is carried out by pyruvate dehydrogenase complex (PDH-complex). Site: - Mitochondria. PDH-complex:- Is a multienzyme complex consisting of 3 enzymes 1. Pyruvate dehydrogenase enzyme. 2. Dihyrdolipoyl-transacetylase enzyme. 3. Dihydrolipoyl Dehydrogenase.  5 coenzymes are needed for the action of PDH-complex: TPP= Thiamin pyrophosphate, Lipoic acid, COASH, FAD and NAD. Summary of oxidative decarboxylation of pyruvate:- Pyruvate DH complex CH3-CO-COOH CH3-CO~SCoA Pyruvate Acetyl CoA COASH CO2 TPP, FAD, Lipoic acid NAD NADH+H+  Regulation of oxidative decarboxylation PDH is regulated by the following 1. Feedback inhibition:- It is inhibited by its end product which is acetyl CoA + NADH+H+ and stimulated by pyruvate + NAD+. 2. Covalent modification:- PDH is present in 2 forms PDH - a = Dephosphorylated form (active form). PDH- b = Phosphorylated form (inactive form). – Phosphorylation is activated by PDH kinase enzyme that is stimulated by increased acetyl CoA/CoA, NADH/NAD or ATP/ADP. – Dephosphoryaltion is activated by PDH phosphatase enzyme that is stimulated by Mg2+, Ca2+ and insulin. 3. Pyruvate DH is inhibited by arsenate or mercuric ions and by thiamin deficiency, leading to accumulation of pyruvic acid in blood and lactic acidosis.  Krebs cycle = Citric Acid Cycle (CAC) Tricarboxylic Acid Cycle (TCA) Definition: - TCA is a series of reactions in which acetyl CoA is completely oxidized into CO2, H2O and energy. Site: - Mitochondria as all enzymes of CAC are present in the mitochondrial matrix except succinate dehydrogenase which is attached to the inner mitochondrial membrane and considered also as one of the components of the respiratory chain. Steps:- See the next page  Energy production by CAC Oxidation of one molecule of acetyl CoA in TCA produces 12 ATP (11 ATP by respiratory chain and 1 by substrate level phosphorylation) as follow;- – 3NADH+H+ by isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase. – 1 FADH2 by succinate dehydrogenase. – 1 ATP by succinate thiokinase (substrate level Phosphorylation). Every NADH+H+ gives 3 ATP while every FADH2 gives only 2 ATP by the respiratory chain. So, Net energy by one cycle of Krebs’ = (3 x 3) + 2 +1 = 12 ATP  Regulation of CAC CAC is the major route for ATP generation and its regulation depends on supply of. 1. Oxidized cofactors e.g NAD+. 2. Availability of ADP. The 3 key enzymes of CAC include citrate synthase, isocitrate DH and α- ketoglutarate DH are regulated as follow; 1. Citrate synthase is – Allosterically inhibited by long chain FAs and competitively inhibited by succinyl CoA. – And stimulated by Ca2+ which increases during muscle contraction (there is increased energy demand). 2. All 3 enzymes are – Stimulated by ADP, NAD and Ca2+. – And inhibited by ATP and NADH+H+. 3. Respiratory control via the respiratory chain and oxidative phosphorylation which ensures oxidation of reduced coenzymes e.g. NADH+H+ and FADH2. 4. Alpha-ketoglutarate DH: as pyruvate DH complex.  Importance of TCA A. Production of energy:- Each molecule of acetyl CoA gives 12 ATP by TCA. B. TCA has amphibolic function i.e it has a catabolic (breakdown) and anabolic (synthetic) functions. – Catabolic function: - Complete oxidation of acetyl CoA derived from CHO, lipid and proteins into CO2 and water. – Anabolic (synthetic) function includes:- 1. Synthesis of non-essential amino acids by transamination. 2. Role of citric acid cycle in gluconeogenesis:-Intermediates as Citrate, α- ketoglutarate and succinyl CoA are glucogenic substance.  3. Succinyl CoA is used in hem synthesis, Ketolysis (breakdown of ketone bodies) and detoxification reactions. 4. Role of CAC in fatty acid and cholesterol synthesis:- C. Production of CO2 which is used in:- 1. Carboxylation of pyruvate to give oxaloacetate that is important for gluconeogenesis. 2. Formation of malonyl CoA during fatty acid synthesis. 3. Formation of carbamoyl phosphate which is used in urea and pyrimidine synthesis. 4. Formation of C6 of purine bases. 5. Synthesis of bicarbonate buffer used in acid base balance.  Inhibitors of CAC in vitro 1. Fluoroacetate inhibits aconitase. 2. Arsenate inhibits α-ketoglutarate DH. 3. Malonate competitively inhibits succinate DH due to structural similarity between malonate and succinic acid.