Paper : 04 Metabolism of carbohydrates Module : 14 Control of citric acid cycle Dr. Vijaya Khader Dr. MC Varadaraj Principal Investigator Dr. S. K. Khare, Professor IIT, Delhi. Paper Coordinator Dr. Ramesh Kothari, Professor UGC-CAS Department of Biosciences Saurashtra University, Rajkot-5, Gujarat-INDIA Dr. S. P. Singh Professor Content Reviewer UGC-CAS Department of Biosciences Saurashtra University, Rajkot-5, Gujarat-INDIA Dr. Vikram Raval, Assistant Professor UGC-CAS Department of Biosciences Content Writer Saurashtra University, Rajkot-5 Gujarat-INDIA 1 Metabolism of Carbohydrates Biochemistry Control of citric acid cycle Description of Module Subject Name Biochemistry Paper Name 04: Metabolism of carbohydrates Module 14: Control of citric acid cycle Name/Title 2 Metabolism of Carbohydrates Biochemistry Control of citric acid cycle CONTROL OF CITRIC ACID CYCLE Objectives 1. To understand basis of citric acid cycle (central metabolic hub) 2. Overview of citric acid cycle and enzymes of TCA cycle 3. Various factors affecting citric acid cycle and control points (regulation) 4. Significance of citric acid cycle 5. Anaplerotic reactions 3 Metabolism of Carbohydrates Biochemistry Control of citric acid cycle To understand basis of citric acid cycle (central metabolic hub) The citric acid cycle is the central hub for metabolic pathways. It is popularly known as tricarboxylic acid cycle (TCA cycle) as well as Kreb’s cycle to honour the inventor of this central metabolic path point For your information (FYI): Sir Hans Adolf Krebs (1900-1981) born on August 25th at Hildesheim, Germany was biochemist and physician who pioneered the study of cellular respiration, and energy production. Globally he is acclaimed for his contributions towards 2 of the important biochemical reactions in the body, citric acid cycle and urea cycle. The former, being the central metabolic reaction for energy generation in cells, is often eponymously known as the "Krebs cycle", fetched him a Nobel Prize in Physiology or Medicine in 1953. Hans Kreb and Hans Kornberg jointly discovered the glyoxylate cycle as well, which is a substitute of TCA cycle observed in plants, bacteria, protists, and fungi. The citric acid cycle is the common terminal point for all metabolic pathways to generate energy from fuel in form of biomolecules such as amino acids, fatty acids as well as carbohydrates In eukaryotes TCA cycle reaction takes place in mitochondria instead glycolysis which occurs in cytoplasm while for prokaryotes both glycolysis and TCA occurs in cytoplasm Here in mitochondria a series of oxidation and reduction reactions occur which results in oxidation of acetyl group of two carbon dioxide molecules while reducing the coenzymes ATP generation takes place. The reduced coenzymes can be reoxidized through electron transport chain clubbed with energy (ATP) generation reaction 4 Metabolism of Carbohydrates Biochemistry Control of citric acid cycle Figure 1: Citric acid cycle: Central metabolic hub Overview of citric acid cycle and enzymes of TCA cycle First a 4- carbon compound (oxaloacetate) fuses with a 2-carbon acetyl moiety to yield a 6-carbon tricarboxylic acid (citrate) through a condensation reaction (aldol condensation) 5 Metabolism of Carbohydrates Biochemistry Control of citric acid cycle Thereafter an oxidative decarboxylation reaction occurs that converts citrate into its isomeric form for removal of a molecule of carbondioxide and formation of 5-carbon compound (α-ketoglutarate) A similar reaction occurs again that selectively converts 5-carbon compound (α- ketoglutarate) into a 4-carbon compound (succinate) by oxidatively decarboxylation Oxaloacetate is later being regenerated from 4-carbon compound succinate by series of reaction through formation of fumarate and malate 2-carbon atoms enter the cycle as an acetyl moiety while 2-carbon atoms leave the cycle in the form of 2 molecules of carbon dioxide Oxygen is required for the citric acid cycle indirectly in as much as it is the electron acceptor at the end of the electron-transport chain, necessary to regenerate NAD+ and FAD 3 hydride ions are being shifted to 3 molecules of NAD+, while a pair of hydrogen atoms is being transferred to 1 molecule of FAD The chief function of the TCA cycle is the harnessing of energy rich electrons from carbon fuel molecules TCA cycle thus provides precursor molecules like amino acids and other reducing agent like NADH, FADH2 that can be used in various other biochemical reactions The citric acid cycle, in conjunction with oxidative phosphorylation, provides the vast majority of energy used by aerobic cells in human beings, greater than 95% The above reactions are catalyzed by at least 8 different enzymes where as the entry of acetyl moiety into the cycle is regulated by the action of a key enzyme called pyruvate dehydrogenase complex (PDHc) These are listed as under along with the substrate and product formed 6 Metabolism of Carbohydrates Biochemistry Control of citric acid cycle Sr. Reaction Enzyme No. * Co-A-SH into acetyl-Co-A (2C) Pyruvate dehydrogenase 1 Citrate (6C) to isocitrate (6C) Aconitase 2 Isocitrate to α- ketoglutarate (5C) Isocitrate dehydrogenase 3 α- ketoglutarate to succinyl-Co-A (4C) α-ketoglutarate dehydrogenase complex 4 Succinyl-Co-A to succinate (4C) Succinyl-Co-A synthase 5 Succinate to fumarate (4C) Succinate dehydrogenase 6 Fumarate to malate (4C) Fumarase 7 Malate to oxaloacetate (4C) Malate dehydrogenase 8 Oxaloacetate (4C) to citrate (6C) Citrate synthase Various factors affecting citric acid cycle control points (regulation) Like glycolysis, TCA cycle too can be regulated at two levels that is at the entry level of substrates and at certain key steps that are catalyzed by enzymes The chief control point of citric acid cycle is believed to be the point of entry of fuel molecule, the acetyl-co-A. Formation of this moiety from various carbohydrate sources acts as major regulatory checkpoint. The reaction is an enzyme catalyzed reaction and pyruvate dehydrogenase enzyme. The activity of this enzyme complex is easily inhibited by ATP, NADH, fatty acids and acetyl-Co-A The PDH complex can be regulated by I. Allosteric modification: PDHc is allosterically inhibited by acetyl-Co-A and NADH while activated by non-acetylated Co-A (Co-A-SH) and NAD+ II. Covalent modification: The PDHc activities are dependent on stages of phosphorylation modulated by specific kinase (PDH kinase) and dephosphorylation catalyzed by discrete phosphatase (PDH phosphatase) 7 Metabolism of Carbohydrates Biochemistry Control of citric acid cycle NADH and acetyl-Co-A act as activator while pyruvate, ADP, Co-A-SH, Ca2+ and Mg2+ act as inhibitor for PDH kinase. On the contrary, the PDH phosphatase, activity is boosted by Mg2+ and Ca2+ Citrate synthase is allosterically inhibited by both ATP as well as long chain fatty acyl-Co- A. ATP, NADH and succinyl-Co-A also act as inhibitor while ADP plays a role of activator Isocitrate dehydrogenase activity is inhibited by NADH and ATP. In constrast it is allosterically activated by ADP, as it enhances affinity towards the substrate α-ketoglutarate dehydrogenase is inhibited by succinyl Co-A, NADH and ATP while activity is stimulated by Ca2+ ions The reactions of citric acid cycle are very well regulated. Majority of the above mentioned enzymes can be regulated in various ways as mentioned in the table below Sr. Enzyme Activator Inhibitor No. AMP, Co-A-SH, Ca+2, ATP, NADH, fatty acids, 1 Pyruvate dehydrogenase NAD+ acetyl-Co-A NADH, citrate, ATP, succinyl- 2 Citrate synthase ADP Co-A 3 Isocitrate dehydrogenase ADP, Ca+2, Mg+2 , Mn+2 ATP 4 α-ketoglutarate dehydrogenase Ca+2 Succinyl-Co-A, NADH + 5 Succinate dehydrogenase FAD, NAD Oxaloacetate, FADH2, NADH Succinate dehydrogenase activity is regulated concentration of oxaloacetate inside the mitochondrial matrix, which in-turn depends on malate dehydrogenase activity Malate dehydrogenase activity heavily depends on the [NADH]/[NAD+] ratio and is regulated by the same 8 Metabolism of Carbohydrates Biochemistry Control of citric acid cycle Significance of citric acid cycle The citric acid cycle is the only a pathway for conversion and interchange of metabolites formed in a cell by various metabolic process of amino acids, fatty acids as well as carbohydrates It mainly provides substrates for various deamination and transamination reactions of amino acid metabolism Since it plays a dual role both as a catalolic and an anabolic process, it is often called an amphibolic pathway Citric acid cycle plays a vital role in energy generation thereby involved in number of catalolic processes. The citric acid cycle is the usual but ultimate pathway for the oxidation of number of intermediates including carbohydrates, lipids, and proteins This is quite regular since glucose, fatty acids, and majority of amino acids are metabolized to form acetyl-Co-A or other intermediates of the TCA cycle The function of the citric acid cycle is said to be vital because it harnesses energy rich electrons from carbon skeleton. 1 acetate unit approximately produces 12 moles of ATP with each cyclic turn The TCA cycle is also regarded to be anabolic in nature as a number of intermediates are produced by it that are capable for biological synthesis/built-up of numerous cellular compounds Citric acid cycle generates intermediates that potentially are involved in gluconeogenesis. Since TCA intermediates forms oxaloacetate, this paves way for gurther decarboxylation and conversion mediated by phosphor-enol-pyruvate carboxy kinase to yield glucose (in liver/kidney) Citric acid cycle also plays major role in biosynthesis of certain essential and non- essential amino acids via reversible transamination reactions. Moreover it also avails the body with carbon skeleton necessary for biosynthesis of amino acids like alanine, 9 Metabolism of Carbohydrates Biochemistry Control of citric acid cycle aspartate, asparagine, glutamate, glutamine, lysine, methionine, theronine and isoleucine Citric acid cycle is also involved in biosynthesis of fatty acid via a key intermediate i.e. Acetyl-Co-A.