CELLULAR RESPIRATION. Chapter 19 & 20. Biochemistry by Campbell and Farell (7 th Edition) By Prof M A Mogale

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1 CELLULAR RESPIRATION Chapter 19 & 20 Biochemistry by Campbell and Farell (7 th Edition) By Prof M A Mogale

2 1. Cellular respiration (energy capture) The enzymatic breakdown of food stuffs in the presence of oxygen to produce cellular energy (ATP)

3 STAGE 1 Conversion of energy-rich food stuffs into acetyl CoA. Different for carbohydrates (glycolysis), proteins and lipids (βoxidation) Very little or no ATP is formed during this stage Energy released is stored as NADH and FADH 2

4 TAGE 2 (Citric acid cycle) Aerobic oxidation of acetyl CoA to CO 2. in mitochondria (8 steps) Common for carbohydrates, amino acids and fatty acids Acetyl CoA (2C) combine with oxaloacetate (4C) to form citric acid (6C) Citric acid is gradually oxidized to regenerate oxaloacetate Energy released is stored as NADH and FADH 2, One mole of ATP is formed by substrate-level phosphorylation

5 The individual reactions of the citric acid cycle Phase 1 : Introduction and loss of two carbon atoms (Steps 1 4) Step 1: Condensation of OA and Acetyl CoA to form citrate and CoA The reaction is catalysed by the enzyme citrate synthase (condensing enzyme) A synthase is an enzyme that make a new covalent bond during the reaction but does not require the direct input of ATP The reaction is an exergonic reaction ΔG o = kj/mol (energy from hydrolysis of the thioester (acetyl CoA))

6 Step 2: Isomerization of Citrate to isocitrate Catalysed by the enzyme aconitase that utilize an Fe sulphur cluster as a cofactor In this reaction a symmetrical (achiral) compound is converted to a chiral compound (a secondary alcohol) yielding several possible isomers The reaction proceed via an enzyme bound cis-aconitate intermediate

7 Aconitase is the target site for the toxic action of fluoroacetate, a plant product that has been used as a pesticide Fluoroacetate is a suicide enzyme (trojan horse) inhibitor which is converted in to the toxic inhibitor fluorocitrate in the active side of the enzyme by the enzymes acetyl CoA synthetase and citrate synthase The action of fluoroacetate is similar to that of the legendary trojan horse

8 One mole of NADH will eventually produce approximately 2,5 mole of ATP when it donates its electron to O 2 during oxidative phosphorylation Step 3: Oxidative decarboxylation of isocitrate to alphaketoglutarate (First oxidation) The reaction is the first of the two oxidative decarboxylation of the citric acid cycle The reaction which proceed in two steps is catalysed by the enzyme isocitrate dehydrogenase One molecule of NADH and one molecule of CO 2 are produced during this stage

9 Step 4: Formation of Succinyl-CoA and CO 2 from alphaketoglutarate (Second oxidation step) The reaction proceed in several steps and is catalysed by a multienzyme system known as alpha-ketoglutarate dehydrogenase complex The alpha-ketoglutarate dehydrogenase enzyme complex utilizes thiamine pyrophosphate(tpp), FAD, lipoic acid and Mg 2+ as enzyme cofactors

10 Step 4: Formation of Succinyl-CoA and CO 2 from alphaketoglutarate (Second oxidation step)

11 The reaction is similar to the one catalysed my pyruvate dehydrogenase complex that convert pyruvate into acetyl CoA. It was initially believed that the two carbon atoms lost as CO 2 in step 3 and 4 of the CA cycle were the acetyl CoA carbons, however current experimental evidence (isotope tracing) show that this carbon atoms come from oxaloacetate The conversion of alpha-ketoglutarate to succinyl CoA is highly exergonic ( G o = kj/mol = -8.0 kcal/mol)

12 Phase 2: Regeneration of oxaloacetate Two carbons have entered the CA cycle as acetyl CoA, and at this stage two have been lost as CO 2 In the remaining reactions, the four carbon intermediate, succinyl-coa is converted to oxaloacetate in four steps (steps 5-8), two of them involving dehydrogenation reactions

13 Step 5: Formation of Succinate (Substrate level phosphorylation) The reaction is catalysed by the enzyme succinyl-coa synthetase A synthetase is an enzyme that creates a new covalent bond and requires a direct input of energy from a compound with a high phosphate transfer potential The free energy of hydrolysis of succinly-coa to produce succinate is kj/mol

14 Thus this reaction, cannot be empowered by hydrolysis of ATP to produce ADP + Pi The energy required for this reaction is provided by the hydrolysis of the thioester bond of succiinyl-coa to produce succinate and CoA-SH In this reaction the energy resulting from thiolysis (forward reaction) is also used to form GTP from GDP Note that the name of the enzyme describe the reverse reaction in which GTP is hydrolysed to produce GDP thereby releasing energy to form the thoester bond This reaction step is referred to as substrate level phosphorylation to distinguish it from formation of ATP coupled to oxidative phosphorylation

15 In mammals, the GTP produced in the succinate synthetase reaction can exchange its terminal phosphoryl group with ADP to yield ATP via a reaction catalysed by the enzyme nucleosite diphosphate kinase

16 Step 6: Flavin-Dependent Dehydrogenation Completion of the cycle involves conversion of the four-carbon succinate to the four-carbon oxaloacetate The first of the remaining reactions is the FAD-dependent dehydrogenation of two saturated carbon atoms to produced a double bond catalysed by succinate dehydrogenase Succinate dehydrogenase is an inner mitochondrial membranebound enzyme that is part of the electron transport chain involved in oxidative phosphorylation

17 The reaction (the oxidation of an alkane to an alkene) is not sufficiently exergonic to reduce NAD, but it does yield enough energy to reduce FAD In general, FAD is a better oxidising agent than NAD + and NADH is a better reducing agent than FADH 2 The action of succinate dehydrogenase is stereo selective, forming only the trans isomer, fumarate

18 Step 7: Hydration of the carbon-carbon double bond Fumarate is converted to L-malate by stereospecific addition of components of a water molecule across the double bond by the enzyme fumarate hydratase (fumarase) This reaction is highly exergonic in the foward direction and has an equilibrium constant of about 4

19 Step 8: Conversion of malate to oxaloacetate This is the final step of the citric acid cycle catalysed by the malate dehydrogenase enzyme The oxidation of alcohols to ketone or aldehyde groups are more energetically favourable and provide sufficient energy to reduce NAD +

20 Stoichiometry of the citric acid cycle The cycle started when a two carbon acetyl-coa combined with a four carbon oxaloacetate to produce citrate Two carbon atoms were removed as carbon dioxide as citrate was further metabolized Four oxidation reactions occurred during the cycle, with NAD + serving as an electron acceptor in three and FAD for the fourth One high energy phosphate was generated by the reaction catalysed by succinyl CoA synthetase Acetyl-CoA + 3NAD + + FAD + GDP + P i + 2H 2 O 2CO 2 + 3NADH +3H + + FADH GTP + CoASH ΔG o = - 40 kj/mol

21 Energetics of the citric acid cycle Although some individual steps in the citric acid cycle may be endergonic the overall reaction of the cycle is exergonic (Table 19.2, C & F, Page 547) ΔG o = - 40 kj/mol In terms of ATP production a total of 9 ATP molecules are produced per one turn of cycle Pathway Substrate-Level Phosphorylation Oxidative Phosphorylation Total ATP Krebs Cycle 1 ATP 3 NADH = 6 ATP 1 FADH 2 = 2 ATP 9 TOTAL 1 ATP 8 ATP 9

22 Regulation of the citric acid cycle Regulation of the citric acid cycle occurs both at the level of entry of fuel in to the cycle and by control of key reactions within the cycle The most important factor controlling the citric acid cycle is the relative intra-mitochondrial concentration of NAD + and NADH Key sites for allosteric regulation are the reactions catalysed by isocitrate degydrogenase and alpha ketoglutarate dehydrogenase

23 Regulation of the citric acid cycle

24 Anaplerosis and Cateplerosis Most citric acid intermediates are used as biosynthetic intermediates and hence may be depleted Anaplerosis is a process whereby citric acid intermediates used in biosynthe tic pathways are replenished Anaplerotic pathways may be classified into three groups, those replenishing oxaloacetate, those replenishing malate and those involving transamination of amino acids Cateplerosis is the oposite of anaplerosis i.e. pathways that drain citric acid intermediates from the cycle for use in biosynthetic pathways

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26 Anaplerotic pathways that replenish oxaloacetate In mammals, the most important anaplerotic pathway for generating oxaloacetate is the reversible ATP-dependent carboxylation of pyruvate to give oxaloacetate This reaction is catalysed by the enzyme pyruvate carboxylase which uses biotin as a cofactor

27 Anaplerosis of malate The anaplerotic pathway for malate involves the malic enzyme (malate dehydrogenase) This enzyme catalyses the reductive carboxylation of pyruvate to give malate

28 Anaplerosis involving transamination of amino acids

29 Cataplerotic Pathways: Role of citric acid in anabolism

30 Role of citric acid cycle in gluconeogenesis

31 Role of citric acid cycle in lipogenesis and pyruvate and pyruvate synthesis

32 The Glyoxylate Cycle: An Anabolic Varient of the Citric Acid Cycle One of the fundamental differences between plant and animal cells is that plants (and some microorganisms) can synthesize carbohydrates (glucose) from fat The conversion of fat into sugars is crucial in the development (germination) of seeds When seed germinate, triacylglycerols are broken down and converted into sugars, a process which provide the energy and raw materials for the growth of the plants Plants synthesize sugars from fats by means of the glyoxylate cycle which is considered as an anabolic variant of the citric acid cycle

33 Glyoxylate then accepts acetate form another cellular acetyl- CoA to produce malate in a reaction catalysed by malate synthase The Glyoxilate Cycle (Cont.) The glyoxylate cycle occurs in the glyoxysome, a specialized plant organelle that caries out both beta-cell oxidation of fatty acids to acetyl CoA and utilization of acetyl CoA in the glyoxylate cycle In the glyoxylate cycle, acetyl CoA (provided by the betaoxidation of fatty acids or by acetate thiokinase) reacts with oxaloacetate to give citrate, which is converted to isocitrate by the enzyme aconitase At this point, the glyoxylate cycle diverges from the citric acid cycle The next reaction is catalysed by isocitrate lyase, which cleaves isocitrate to glyoxylate and succinate

34 The glyoxylate cycle

35 The Link between the Citric Acid cycle and molecular Oxygen The citric acid cycle does not operate under anaerobic conditions This is because of the fact that the citric acid cycle is regulated among other things, by the concentration of NADH produced by the cycle After being produced by the citric acid cycle, NADH (and FADH2) donates its electrons to molecular oxygen through the respiratory chain Thus, in the absence of oxygen NADH will accumulate and inhibit the citric acid cycle