Description of Module Paper : 04 Metabolism of carbohydrates Module : Citric acid cycle Dr. Vijaya Khader Dr. MC Varadaraj Content Reviewer: Dr. Ramesh Kothari, Professor Principal Investigator, UGC-CAS Department of Biosciences Saurashtra University, Rajkot-5 Paper Coordinator and Gujarat-INDIA Content Writer Prof. S. P. Singh, Professor UGC-CAS Department of Biosciences Saurashtra University, Rajkot-5 Gujarat-INDIA 1 Metabolism of Carbohydrates Biochemistry Citric Acid Cycle Subject Name Biochemistry Paper Name 04 Metabolism of carbohydrates Module Citric acid cycle Name/Title Objectives 1. History and introduction of citric acid cycle 2. Conversion of pyruvate to activated acetate by pyruvate dehydrogenase 3. Explain Reactions of citric acid cycle 4. Amphibolic nature of Citric acid cycle 2 Metabolism of Carbohydrates Biochemistry Citric Acid Cycle 1. OVERVIEW Citric acid cycle is also called Tricarboxylic acid (TCA) cycle or Krebs cycle is a sequence of biochemical reactions that occurs in all aerobic organisms for energy generation. Energy is generation is carried out by the oxidation of acetate, which is derived from carbohydrates, lipids and proteins converted into Co2 and chemical energy stored in the form of adenosine triphosphate (ATP). Furthermore the TCA cycle supplies precursors 3 Metabolism of Carbohydrates Biochemistry Citric Acid Cycle for synthesis of several amino acids and reducing agent such as NADH, which involves in various other biochemical reactions. TCA cycle is one of the initially established mechanism of cellular metabolism suggested by the central importance in various biochemical pathways. The name of this biochemical pathway is derived from tricarboxylic acid (e.g. citric acid). Citric acid is first utilized and then regenerated by this sequential reactions to complete the cycle. The major function of these two closely associated pathways is the oxidative breakdown of nutrients into production of usable energy in the form of ATP. In eukaryotic cells, the Krebs cycle occurs in the mitochondrial matrix. In prokaryotic cells the TCA reaction occurs in the cytosol through the proton gradient for energy generation. In 1935 Albert Szent-Gyorgyi showed that Succinate Fumarate Malate Oxaloacetate Carl Martius and Franz Knoop showed Citrate cis-aconitate Isocitrate α ketoglutarate Succinate Fumarate Malate Oxaloacetate - Overall reaction of the citric acid cycle is: + 3NAD + FAD + GDP + Pi + acetyl-CoA → 3NADH + FADH2 + GTP + CoA + 2CO2 from glucose: + Glucose + 2NAD + 2ADP + 2Pi → 2pyruvate + 2NADH + 2ATP 4 Metabolism of Carbohydrates Biochemistry Citric Acid Cycle + 2pyruvate + 2NAD + 2CoA → 2acetyl-CoA + 2NADH + 2CO2 + 2acetyl-CoA + 6NAD + 2FAD + 2GDP + 2Pi → 6NADH + 2FADH2 + 2GTP + 2CoA + 4CO2 2GTP + 2ADP → 2ATP + 2GDP__________________________________________ + Glucose + 10NAD + 4ADP + 4Pi + 2FAD → 10NADH + 2FADH2 + 4ATP + 6CO2 → 30ATP + 4ATP + 4ATP = 38ATP 5 Metabolism of Carbohydrates Biochemistry Citric Acid Cycle Overall reactions of citric acid cycle 6 Metabolism of Carbohydrates Biochemistry Citric Acid Cycle 2. Conversion of pyruvate to activated acetate by pyruvate dehydrogenase - Pyruvate converts into the acetyl-CoA before enters into the TCA. - The coenzyme A is act as a carrier for acetyl and other acyl group. - Acetyl-CoA is a “high-energy” compound. 7 Metabolism of Carbohydrates Biochemistry Citric Acid Cycle A. Pyruvate dehydrogenase is a multienzyme complex - By the oxidative decarboxylation process Acetyl-CoA is formed from pyruvate using multienzyme complex named as a pyruvate dehydrogenase. + Pyruvate + CoA + NAD → acetyl-CoA + CO2 + NADH - Pyruvate dehydrogenase a multienzyme complex consists of: 1. Pyruvate dehydrogenase (E1) 2. Dihydrolipoyl transacetylase (E2) 3. Dihydrolipoyl dehydrogenase (E3) Figure: Conversion of Pyruvate to Acetyl-CoA by Pyruvate dehydrogenase multienzyme complex 8 Metabolism of Carbohydrates Biochemistry Citric Acid Cycle B. Control of pyruvate dehydrogenase Product inhibition - When the relative concentrations of NADH and acetyl-CoA are high, the reversible reactions catalyzed by E2 and E3 are driven backwards. Therefore formation of acetyl- CoA is inhibited. - Thus the E2 and E3 activities are controlled by product inhibition (acetyl-CoA for E2 and NADH for E3). Covalent modification (Eukaryotic complex only) E1 is regulated by phosphorylation/dephosphorylation. When the Ser of E1 is phosphorylated, the enzyme is inactivated. 9 Metabolism of Carbohydrates Biochemistry Citric Acid Cycle Insulin activates Activators of phosphatase: Mg2+, Ca2+ Activators of kinase: Acetyl-CoA, NADH Inhibitors of kinase: Pyruvate, ADP, Ca2+, high Mg2+, K+ Remember: Insulin inhibits phosphorylation and activates dephosphorylation in order to reduce the (glucose) in blood at the starting point of glycolysis. - Now, insulin also works to reduce the end product of glycolysis, i.e., activates dephosphorylation of E1 to convert pyruvate to acetyl-CoA. - Acetyl-CoA is not only the fuel of citric acid cycle, but also the precursor of fatty acids. 10 Metabolism of Carbohydrates Biochemistry Citric Acid Cycle 3. Reactions of the citric acid cycle A. Citrate is formed from Oxaloacetate and Acetyl Coenzyme A by citrate synthase enzyme The citric acid cycle initiates through the condensation of an oxaloacetate (four-carbon unit), and the acetyl group of acetyl CoA (a two-carbon unit). Oxaloacetate reacts with acetyl CoA and H2O to yield as citrate and CoA. B. Isomerization of Citrate into Isocitrate In the citrate molecule the tertiary hydroxyl group is not properly situated for the oxidative decarboxylations that follow. Therefore, isomerization occurs of citrate into isocitrate to allow the six-carbon component to undergo oxidative decarboxylation. The isomerization of citrate is accomplished by a dehydration reaction following a hydration reaction. The result is a substitution of a hydrogen atom and a OH- group. Both the steps are catalyzed by the enzyme aconitase because cis-aconitate is an intermediate. ∆G°’ = -13.3 kJ/mol 11 Metabolism of Carbohydrates Biochemistry Citric Acid Cycle Fluorocitrate inhibits aconitase - Fluoroacetate, one of the most toxic small molecules (LD50 = 0.2 mg/kg), is converted to (2R,3R)-fluorocitrate, which specifically inhibits aconitase since Ser-642 cannot remove the proton at C2. Less acidic Less toxic Very toxic C. Oxidation and decarboxylation of isocitrate to a-Ketoglutarate The isocitrate is oxidized and decarboxylated by enzyme isocitrate dehydrogenase. Oxalosuccinate act as an intermediate in this reaction. ∆G°’ = -20.9 kJ/mol - There are two isozymes in mammalian cells. 12 Metabolism of Carbohydrates Biochemistry Citric Acid Cycle 1. NAD+-dependent form is in mitochondria and requires Mn2+ or Mg2+. 2. NADP+-dependent form is in both cytosol and mitochondria. D. The oxidative decarboxylation of α- Ketoglutarate to forms Succinyl CoA Catalyzes the oxidative decarboxylation of an α-keto acid, releasing CO2, forming succinyl- CoA and reducing NAD+ to NADH - A α-Ketoglutarate dehydrogenase that consists of α-ketoglutarate dehydrogenase (E1), dihydrolipoyl transsuccinylase (E2), and dihydrolipoyl dehydrogenase (E3). - The overall reaction closely resembles that are catalyzed by the pyruvate dehydrogenase multienzyme complex, i.e., 1. Decarboxylation -----------------------E1 2. Succinyl group transfer -----------E2 3. Succinyl-CoA formation. -------- E2 4. Oxidation of E2. ------------------- E3 + 5. Reduction of NAD . ---------------E3 E. Succinate formed from succinyl-CoA - Hydrolysis of “high-energy” compound succinyl-CoA is coupled with the production of a “high- energy” nucleosidetriphosphate (GTP). 13 Metabolism of Carbohydrates Biochemistry Citric Acid Cycle - The thioester bond energy of succinyl-CoA is conserved through the formation of a series of “high-energy” phosphate (~Pi). The succinate formation is as follows: - GTP is converted into ATP by nucleoside diphosphate kinase. GTP + ADP ↔ GDP + ATP ∆G°’ = 0 kJ/mol F. Fumarate is formed from Succinate - Stereospecific dehydrogenation occurs of succinate to fumarate and produces FADH2. ∆G°’ = 0 kJ/mol - The FAD is covalently bound to the succinate dehydrogenase enzyme. Thus, FADH2 cannot be oxidized as a cofactor. FADH2 is oxidized by the electron transport chain reaction. 14 Metabolism of Carbohydrates Biochemistry Citric Acid Cycle - For the reason, succinate dehydrogenase is the only membrane-bound enzyme of citric acid cycle. The others are dissolved in the mitochondrial matrix. - The enzyme is sturdily inhibited by malonate (structural analog of succinate). G. Malate formed from fumarate by hydrogenation - Hydrogenation occurs of fumarate’s double bond to form L-malate. ∆G°’ = -3.8 kJ/mol H. Oxaloacetate regenerates from Malate - Oxaloacetate regenerates by the oxidation of hydroxyl group of L-malate to ketone in a NAD+-dependent reaction,. ∆G°’ = 29.7 kJ/mol - This reaction is relatively high endergonic reaction (∆G˃0) I. Integration of the citric acid cycle - Following chemical transformations occurs in Citric acid cycle. 1. One acetyl group (-COCH3) → 2CO2 (4-electron pair process). O S C + - CoA CH3 + 3H2O 2CO2 + CoA--SH + 8H + 8e 2. Reduction of three NAD+ to three NADH (3-electron pairs process) and equivalent to 15 Metabolism of Carbohydrates Biochemistry Citric Acid Cycle + + - + 9ATP generation, i.e., 3NAD + 6H + 6e → 3NADH + 3H 3. Reduction of one FAD to FADH2 (1-electron pairs process) and equivalent to 2ATP + - generation, i.e., FAD + 2H + 2e → FADH2 4. Generation of