Carbohydrate Metabolism Tricarboxylic Acid Cycle 1

Objectives Define Cellular Respiration Describe the reactions of TCA cycle and the role of the pathway in metabolism Recognize the importance of Acetyl CoA as the end-product of degradative pathways 2

Cellular Respiration Cellular Respiration – molecular processes by which cells consume O2 and produce CO2 Cellular respiration occurs in three stages Organic fuels – glucose, fatty acids, and amino acids – are oxidized to form the two-carbon compound, acetyl CoA Acetyl group metabolized via the Krebs Cycle and enzymatic ally oxidized to CO2. Energy liberated trapped in coenzymes, NADH and FADH2 Oxidation of these coenzymes liberate protons and electrons which are finally transferred to O2 via the electron-transport chain (respiratory chain). Process liberates large amount of energy and conserved in the form of ATP by process known as oxidative phosphorylation. 3

Energy Production in Metabolism (Summary of Oxidation of Foodstuffs in Three Stages) Secondary (Intermediary Metabolism Tertiary Metabolism (ETC) Primary metabolism (Digestion) Acetyl CoA ATP Carbohydrates  Glucose TCA Cycle Energy Lipids  Fatty acids ATP CO2 Proteins  Amino acids NADH FADH2 H2O Oxidation of these coenzymes liberate protons and electrons which are finally transferred to O2 via the electron-transport chain (respiratory chain). O2 TCA Cycle 4

Amphibolic - acts both catabolically and anabolically The Citric acid cycle It is called the Krebs cycle or the tricarboxylic and is the “hub” of the metabolic system. It accounts for the majority of carbohydrate, fatty acid and amino acid oxidation. It also accounts for a majority of the generation of these compounds and others as well. Amphibolic - acts both catabolically and anabolically

TCA Cycle - Pathway characteristics 1. Cyclic pathway consisting of 8 enzyme-mediated steps 2. Central to most of metabolism 3. Strictly an aerobic process Only works under aerobic conditions because NAD+ and FAD must be regenerated by oxidative phosphorylation. Therefore, absence (anoxia) or partial deficiency (hypoxia) of O2 causes total or partial inhibition of the cycle. 4. Occurs in mitochondrial matrix of eukaryotes. Responsible for the conversion of acetyl-CoA to 2 CO2. while conserving the free energy for ATP production. The energy is stored as: NADH (3ATP), 1 FADH2 (2 ATP), 1 GTP (1 ATP) for each molecule of acetyl CoA oxidized 6

Sources of Acetyl CoA Acetyl CoA Glycogen Glycogenolysis Glucose (Acetyl CoA = CH3-CO~S-CoA) Coenzyme A, abbreviated as CoA or CoASH, is composed of β-mercaptoethylamine, pantothenic acid (a vitamin) and adenosine 3’-phosphate 5’-diphosphate Glycogen Glycogenolysis Glucose Glycolysis Amino Acids Fatty Acids Pyruvate Acetyl CoA 7

CoA acts as a carrier of acetyl groups Acetyl-CoA is a “high energy” compound: The DG' for the hydrolysis of its thioester is -31.5 kJ• mol-1 making it greater than the hydrolysis of ATP Pyruvate dehydrogenase converts pyruvate to acetyl-CoA and CO2

Formation of Acetyl CoA from Pyruvate Pyruvate Dehydrogenase (PDH) Electron Transport System Pyruvate   CoASH   FAD NADH + H+     CO2 FADH2 NAD+ PDH not part of TCA cycle but important for supply of acetyl CoA Acetyl CoA 9

The citric acid cycle enzymes are found in the matrix of the mitochondria Substrates have to flow across the outer and inner parts of the mitochondria

Irreversible The fate of carbon in the TCA cycle Citrate synthase Succinαte dehydrogenase aconitase Irreversible Fumarate aconitase Isocitrate dehydrogenase Succinαte thiokinase α ketoglutarate dehydrogenase 11

Reactions of the TCA cycle The cycle comprises the condensation of a molecule of acetyl-CoA (2C) with a 4C dicarboxylic acid (oxaloacetate), to form a 6C tricarboxylic acid (citrate). Then a series of reactions follows where two molecules of CO2 are released and oxaloacetate is regenerated Pathway involves 3 irreversible reactions. 1. Formation of citrate Citrate synthase catalyzes the irreversible condensation of acetyl-CoA with oxaloacetate (OAA) to form citrate. 13

2. Formation of isocitrate through cis-aconitate In the next step, citrate is reversibly isomerised to isocitrate by aconitase. This reaction is a two- step process. In the first step, cis-aconitate is formed and then finally converted to isocitrate 14

3. Formation of α-ketoglutarate Isocitrate is oxidatively decarboxylated to α-ketoglutarate by isocitrate dehydrogenase (IDH). Reaction is irreversible with the intermediate formation of oxalosuccinate which undergoes spontaneous irreversible decarboxylation to form α-ketoglutarate. NADH is produced. 15

4. Formation of succinyl CoA α-Ketoglutarate is oxidatively decarboxylated to succinyl-CoA by the α-ketoglutarate dehydrogenase complex. Irreversible reaction. NADH and CO2 are generated. This multienzyme complex is very similar to the PDH complex in terms of reaction catalyzed and coenzyme requirements. Succinyl-CoA and α-ketoglutarate are important entry and exit points of C atoms from the TCA cycle and amino acid metabolism. 16

5. Generation of succinate The conversion of succinyl-CoA to succinate by succinyl CoA synthetase (or succinate thiokinase) involves use of the high-energy thioester of succinyl-CoA to drive synthesis of a high-energy nucleoside phosphate - GTP (substrate-level phosphorylation). Mitochondrial nucleoside diphosphokinase phosphorylates ADP, producing ATP and regenerates GDP for the continued operation of succinyl CoA synthetase. Nucleoside diphosphokinase catalyses GTP + ADP ↔ ATP +GDP 17

Reaction competitively inhibited by malonate. 6. Formation of fumarate Succinate dehydrogenase catalyzes the oxidation of succinate to fumarate. Reaction competitively inhibited by malonate. The H atoms are accepted by FAD. The FADH2 then enters into ETC to generate two ATP. The enzyme is a flavoprotein. 7. Formation of malate The formation of malate from fumarate is catalysed by fumarase (fumarate hydratase) 18

8. Regeneration of Oxaloacetate Malate dehydrogenase (MDH), the final enzyme of the TCA cycle which converts malate to oxaloacetate The forward reaction of the cycle, the oxidation of malate yields oxaloacetate (OAA). The NADH generated in this step enters ETC to produce three ATP 19

ATP Yield Energy yield Citrate  -KGA  NADH 3xATP -KGA  Succinyl CoA  NADH 3xATP Succinyl CoA  Succinate (1xGTP) 1xATP Succinate  Fumarate  FADH2 2xATP Malate  OAA  NADH 3xATP Total (Acetate to CO2) 12xATP Pyruvate  Acetyl CoA  NADH 3xATP Total (Pyruvate to CO2) 15xATP 20

Inhibitors of TCA Cycle Arsenite is an inhibitor of pyruvate DH, -ketoglutarate DH Arsenite binds to thiol group (present in enzyme)  inhibition of enzyme  Inhibition of pathway due to Arsenic poisoning Thiol (sulphydryl) group ( CoASH, lipoamide) involved in catalytic action of pyruvate DH and also -ketoglutarate DH Fluoroacetate is an inhibitor of aconitase Found in leaves of various poisonous plants in S. Africa O  C  S  CoA  CH2F  Fluoroacetyl CoA CH2COO-  HO  C  COO- CH2FCOO- Fluorocitrate CoASH COO-  CH2F Fluoroacetate CoASH OAA Fluorocitrate is a potent inhibitor of aconitase, enzyme involved in the conversion of citrate to isocitrate – hence inhibits operation of TCA cycle. 22

Inhibition of TCA Cycle By Malonate Malonate is an inhibitor of succinate DH Malonate is a structural analogue of succinate COO-  CH2 Malonate COO-  CH2  Succinate Malonate strongly inhibits succinate DH, an example of competitive inhibition 23

Biological role or Significance of TCA Cycle 1. Final common oxidative pathway for all the major ingredients of foodstuffs. Carbohydrates converted to acetyl CoA. Fatty acids are broken down to acetyl CoA Amino acids enter into the cycle into some points or other. 2. Oxidation of fatty acids (which yield acetyl CoA) needs the help of oxaloacetate. OAA acts as a “carrier”; it enters into the cycle, but is regenerated in the end. The major source of oxaloacetate is pyruvate (formed from carbohydrate). Hence, carbohydrates are absolutely required for oxidation of fats, or fats are burned under the fire of carbohydrates. Hence no net synthesis of carbohydrates from fats. Carbohydrates Fats Fat Carbohydrates 3. All amino acids after transamination enter into some or other points of the pathway E.g. glutamic acid enters at the level of α-ketoglutarate; aspartate enters at the level of oxaloacetate. 24

4. Amino acid metabolism ends in TCA cycle Some amino acids such as leucine, catabolized to acetyl CoA , completely oxidised, or channeled to ketone body formation. Hence they are called as ketogenic amino acids. 5. Amphibolic Pathway TCA cycle is truly amphibolic (catabolic + anabolic). It is amphipathic in nature. There is a continuous influx (pouring into) and a continuous efflux (removal) of 4-carbon units from the TCA cycle. 6. Anaplerotic role of TCA cycle TCA cycle as a source of precursors of biosynthetic pathways, heme is synthesized from succinyl CoA and aspartate from oxaloacetate. There is a continuous efflux of 4-carbon units from the cycle. To counter balance this loss, and to keep concentrations of the 4-carbon units in the cell, anaplerotic reactions are essential. This is called as anaplerotic role of TCA cycle (ana-up, pleritokos- to fill) Anaplerotic reactions are ‘filling up’ reactions or ‘influx’ reactions which supply 4- carbon units to the TCA cycle. 25

Amphibolic Nature of TCA Cycle TCA Cycle degrades acetate to CO2 and hence catabolic However, intermediates are also tapped off from the cycle for synthesis of various products, hence anabolic Hence TCA is amphibolic i.e. has both catabolic and anabolic functions If intermediates not replaced, cycle will come to a halt. Pathways which replenish the intermediates are known as anaplerotic reactions and main one is pyruvate carboxylase Catabolic reactions Pyruvate Acetyl CoA Citrate -KGA Succinyl CoA Fumarate Oxaloacetate CO2 CO2 Anabolic reactions Glucose Anaplerotic reactions Fatty acids cholesterol Amino acids Amino acids Porphyrins 26 Isoleucine, methionine, valine Odd chain fatty acids

At the end of the lecture, students should be able Learning outcomes: At the end of the lecture, students should be able Outline the TCA pathway and give the irreversible reactions in the pathway Give the reactions which lead to the degradation of acetyl CoA, resulting in the liberation of CO2 Describe the steps which result in the formation of reducing equivalents Discuss the importance of the pathway in energy production and biosynthesis Show the yield of ATP in each turn of the cycle and when glucose is completely degraded to CO2 27

Overview