Under aerobic conditions, pyruvate enters the mitochondria where it is converted into acetyl CoA.

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Sharlene Vivian Berry
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2 Under aerobic conditions, pyruvate enters the mitochondria where it is converted into acetyl CoA. Acetyl CoA is the fuel for the citric acid cycle, which processes the two carbon acetyl unit to two molecules of CO 2 while generating high-energy electrons that can be used to form ATP.

4 A key function of the citric acid cycle is to harvest highenergy electrons in the form of NADH and FADH 2. The two carbon acetyl unit from acetyl CoA condenses with oxaloacetate to form citrate, which is subsequently oxidized. The high-energy electrons are used to reduce O 2 to H 2 O. This reduction generates a proton gradient that is used to synthesize ATP.

6 The citric acid cycle itself does not generate a large amount of ATP, instead it removes electrons from Acetyl CoA forming NADH and FADH 2. These electron carriers yield nine ATP molecules when oxidized by oxidative phosphorylation. Electrons released in the re-oxidation of NADH and FADH 2 flow through a series of membrane proteins to generate a proton gradient across the mitochondrial membrane. These protons then flow through ATP synthase to generate ATP from ADP. The citric acid cycle, in conjunction with oxidative phosphorylation provide the vast majority of energy used by aerobic cells (>90% in human beings)

8 The Pyruvate Dehydrogenase Complex Links Glycolysis to the Citric Acid Cycle and is irreversible: Pyruvate dehydrogenase links glycolysis to the citric acid cycle

9 Electron micrograph of the giant pyruvate dehydrogenase complexes in E. coli

10 The pyruvate dehydrogenase consists of three distinct enzymes:

11 The reaction requires the three enzymes of pyruvate dehydrogenase and five coenzymes; thiamine pyrophosphate (TPP), lipoic acid and FAD serve as catalytic cofactors:

12 The conversion of pyruvate to acetyl CoA consists of three steps: decarboxlyation, oxidation and transfer of the acetyl group to CoA These three steps are coupled in the enzyme complex

13 Decarboxylation: pyruvate combines with TPP to yield hydroxyethyl-tpp this is catalyzed by the E1enzyme of the complex

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15 Oxidation: the hydroxyethyl group attached to TPP is oxidized to form an acetyl group while being simultaneously transferred to lipoamide

16 Formation of Acetyl CoA: The acetyl group is transferred from acetyllipoamide to CoA to form acetyl CoA: Dihydrolipoyl transferase (E2) catalyzes this reaction

17 To complete another catalytic cycle the dihydrolipoamide is oxidized to lipoamide: In this fourth step the oxidized form of lipoamide is regenerated by dihydrolipoyl dehydrogenase (E3)

18 Pyruvate dehydrogenase complex

19 E2 (trimer) has the reactive disulfide bond of Lipoamide tethered through an e-n of lysine

20 E2 (a3)

21 Reactions of the pyruvate dehydrogenase complex: 1) Decarboxylation, (2) insertion of lipoamide arm into E1, (3) E1 catalyzes the transfer of the acetyl group to lipoamide, (4) the acetyl moiety is transferred to CoA, (5) E3 oxidized lipoamide by FAD, (6) NADH is produced by the re-oxidation of FADH 2

22 Citrate synthase catalyzes the condensation of acetyl CoA and oxaloacetate to form citrate.

23 The first enzyme of the Citric Acid Cycle Citrate synthetase forms citrate from oxaloacetate and acetyl CoA: (condensation reaction)

24 Citrate synthase is first bound by oxaloacetate resulting in a significant conformational change generating a binding site for acetyl CoA (homo-dimer)

26 Aconitase catalyzes the isomerization of citrate into isocitrate so that the 6-carbon unit can undergo oxidative decarboxylation: Aconitase is a non-heme iron containing enzyme the Fe-S cluster participates in the dehydration and rehydration of the substrate

27 Binding of citrate to the Fe-S complex of aconitase: only one atom of Fe is directly involved:

28 Isocitrate is oxidized and decarboxylated to a-ketoglutarate by the enzyme isocitrate dehydrogenase: This is the first of four oxidative reduction reactions of the citric acid cycle generating NADH

29 Succinyl coenzyme A is formed by the oxidative decarboxylation of a-ketoglutarate this reaction is catalyzed by the enzyme a- ketoglutarate dehydrogenase complex: a-ketoglutarate dehydrogenase complex consists of three kinds of enzymes and is homologous to the pyruvate dehydrogenase complex

30 Succinyl CoA synthetase catalyzes the cleavage of a thioester linkage and concomitantly forms ATP. The formation of ATP by succinyl CoA synthetase is an example of a substrate-level phosphorylation because succinyl phosphate, a high phosphoryl-transfer potential compound, donates a phosphate to ADP.

31 Succinyl CoA synthetase (aka: succinate thiokinase) both ATP and GTP can be formed from their diphospho-counterparts: ADP ATP

32 The succinyl coenzyme A synthetase-catalyzed reaction occurs in 4 steps: 1. Phosphate attacks succinyl CoA, displacing the CoA and forming succinyl phosphate, a molecule with high phosphoryl-transfer potential. 2. Histidine removes the phosphoryl group, forming phosphohistidine and succinate. 3. The phosphohistidine reorients to approach the bound ADP. 4. The phosphoryl group is transferred to ADP to form ATP.

33 Reaction mechanism of succinyl CoA synthetase: 1) Orthophosphate displaces CoA, (2) histidine residue removes the phosphate resulting in the release of succinate, (3)the phospho histidine residue swings over to the bound ADP and (4) the phosphoryl group is transferred to the nucleoside diphosphate forming the triphosphate ADP ATP

34 Structure of succinyl CoA synthetase

35 Oxaloacetate is regenerated by the oxidation of succinate: Succinate dehydrogenase Fumarase Oxidation-hydrationoxidation a common metabolic motif Malate dehydrogenase

36 Fumarase catalyzes a stereospecific trans addition of H + and OH - so that only the L-isomer of malate is formed

37 Citric Acid Cycle

38 The net reaction of the citric acid cycle is: The electrons from NADH will generate 2.5 ATP when used to reduce oxygen in the electron-transport chain. The electrons from FADH 2 will power the synthesis of 1.5 ATP with the reduction of oxygen in the electron-transport chain.

40 ATP

41 The formation of acetyl CoA from pyruvate is irreversible in animal cells. Acetyl CoA has two principle fates: metabolism by the citric acid cycle or incorporation into fatty acids.

42 The formation of Acetyl CoA by pyruvate dehydrogenase is irreversible and therefore regulated

43 Enzyme E 1 is a key site of regulation. A kinase associated with the complex phosphorylates and inactivates E 1. A phosphatase, also associated with the complex, removes the phosphate and thereby activates the enzyme. The pyruvate dehydrogenase complex is also regulated by energy charge. ATP, acetyl CoA, and NADH inhibit the complex. ADP and pyruvate stimulate the complex.

46 The citric acid cycle is controlled at several points The rate of the citric acid cycle is precisely adjusted to meet the ATP needs in an animal cell. The two primary control points are the allosteric enzymes; isocitrate dehydrogenase and a-ketoglutarate dehydrogenase These are the first two enzymes in the cycle to generate highenergy electrons

47 PDH ID akgd

48 The first control point isocitrate dehydrogenase is allosterically stimulated by ADP, which enhances the enzyme s affinity for substrates. The binding of substrate, NAD +, Mg + and ADP are mutually cooperative. In contrast ATP is inhibitory as is binding of the reaction product NADH which inhibits isocitrate dehydrogenase by displacing NAD +. Note that several steps in the cycle require NAD + or FAD + which are abundant only when the energy charge is low.

49 PDH ID akgd

50 The second control site in the citric acid cycle is a-ketoglutarate dehydrogenase. Some aspects of this enzyme s control are like those of the pyruvate dehydrogenase complex, as might be expected by the homology of the reactions catalyzed by these two enzymes. a-ketoglutarate dehydrogenase is inhibited by the products of the reaction succinyl CoA and NADH. In addition a-ketoglutarate dehydrogenase is inhibited by elevated levels of ATP.

51 The use of isocitrate dehydrogenase and a-ketoglutarate dehydrogenase as control points integrate the citric acid cycle with other metabolic pathways. The inhibition of isocitrate dehydrogenase leads to a build up citrate because the inter-conversion of isocitrate and citrate readily occurs. Citrate can be transported to the cytoplasm where it signals phosphofructokinase to reduce glycolysis.

52 If energy needs are met and ATP levels high the components of the citric acid cycle can be used in other metabolic pathways:

53 mitochondrial enzyme Pyruvate dehydrogenase complex Low ATP levels favor active pathways to produce energy

54 Hat makers of the past used HgNO 3 to soften the firs used in hat making. The Hg diffuses through the skin inhibiting the E2 enzyme of pyruvate dehydrogenase through the dihydrolipoyl groups. This causes a neurological disorder; hence the term Mad-Hatter

55 Neuronal cells are particularly susceptible to inhibitors of pyruvate dehydrogenase because they can only use glucose as a source of fuel. Other cell types can break down fats as a source of fuel for the citric acid cycle.

56 Developed during WW I as an antidote to lewisite an arsenic based chemical weapon (gas) BAL (British anti-lewisite) it functions to chelate the arsenite removing it from the cell:

57 Defects in the citric acid cycle contribute to the development of cancer Three enzymes are involved: succinate dehydrogenase, fumarase and pyruvatedehydrogenase kinase Hypoxia inducible factor 1 (HIF-1) is a transcription factor that is normally unstable however when stabilized by hypoxia (low O 2 concentration) HIF-1 can now activate genes involved in glycolysis. Defects in the above three enzymes can result in the accumulation of succinate and fumarate in the mitochondria allowing accumulation in the cytoplasm. In the cytoplasm these substrates inhibit the enzyme required to lead to the degradation of HIF-1 (prolyl hydrolase 2) and glycolysis is favored by the action of HIF-1.

Citric Acid Cycle: Central Role in Catabolism. Entry of Pyruvate into the TCA cycle

Citric Acid Cycle: Central Role in Catabolism Stage II of catabolism involves the conversion of carbohydrates, fats and aminoacids into acetylcoa In aerobic organisms, citric acid cycle makes up the final