CITRIC ACID CYCLE 1 Oxidation of Pyruvate under Aerobic Conditions: • The Oxidation of Pyruvate to Acetyl-CoA is the Irreversible Route from Glycolysis to the Citric Acid Cycle • Pyruvate, formed in the cytosol, is transported into the mitochondrion by a proton symporter. Inside the mitochondrion, pyruvate is oxidatively decarboxylated to acetyl-CoA by a multienzyme complex that is associated with the inner mitochondrial membrane, known as Pyruvate Dehydrogenase (PDH). 2 • PDH is an Irreversible Mitochondrial Enzyme. PDH Composed of Three Enzymes & Five Coenzymes. • PDH & all f the -ketoacid dehydrogenase complexes are enzyme complexes composed of multiple subunits of three different enzymes, E1, E2, and E3. 3 • E1 is an -ketoacid decarboxylase or Dehydrogenase contains thiamine pyrophosphate (TPP) which catalyzes the Decarboxylation of the carboxyl group of the -keto acid. • E2 is a transacylase containing lipoate; it transfers the acyl portion of the -keto acid from thiamine to CoASH. • E3 is dihydrolipoyl dehydrogenase, transfers the electrons from reduced lipoate to its tightly bound FAD molecule, thereby oxidizing lipoate back to its original disulfide form. 4 Pyruvate Dehydrogenase Complex 5 Coenzymes of Pyruvate Dehydrogenase Complex •Thiamin Pyrophosphate (TPP) in the PDH: • The acyl portion of the -keto acid is transferred by TPP on E1 to lipoate on E2. Then, E2 transfers the acyl group from lipoate to CoASH. • This process has reduced the lipoyl disulfide bond to sulfhydryl groups (dihydrolipoyl). 6 Lipoic acid & Transacylase Enzyme •Lipoate is attached to the transacylase enzyme (E2) through its carboxyl group & the terminal -NH2 of a lysine of the E2. •The oxidized lipoate disulfide form is reduced as it accepts the acyl group from TPP attached to E1. •Lipoic picks up the acyl fragment from thiamine and transfer it to the active site containing bound CoASH. 7 8 Coenzyme A (CoASH) • E2 transfers the acyl group from lipoate to CoASH with reduction of the lipoyl disulfide to sulfhydryl group (dihydrolipoyl). SH of mercaptoethylamine forms a thioester high energy bond with acetate to form AcetylCoA. 9 FAD (Flavine adenine dinucleotides) • Composed of Adenine dinucleotide + Ribitol +Isoalloxazine. • -SH gp of Dihydrolipoic acid is oxidized by FAD (prosthetic gp of E3) + • Nitrogens 1& 5 of the Isoalloxazine ring carry the 2 H in FADH2. 10 (5) NAD (Nicotinamide Adenine Dinucleotide) • FADH2 of E3 donates its 2 ele & H- to NAD. PDH is now ready for another catalytic cycle. 11 Regulation of Pyruvate Dehydrogenase complex (PDH) • 1) PDH kinase, phosphorylates PDH converting it to an inactive form. The PDH kinase is inhibited by ADP & Pyruvate. • 2) PDH phosphatase, removes the phosphate, activating PDH. • The phosphatase is activated by Ca2+. In heart, increased intramitochondrial Ca2+ during contraction activates the phosphatase, thereby increasing the amount of active, nonphosphorylated PDH. • 3) Pyruvate & CoASH, when are bound to PDH, the kinase activity is inhibited & PDH is active. 12 Pyruvate Dehydrogenase Regulated by Covalent Modification 13 4) Pyruvate Dehydrogenase Is Regulated by End-Product Inhibition & Covalent Modification. • Acetyl CoA & NADH, when these products bind to PDH, the kinase activity is stimulated, and the enzyme is phosphorylated to the inactive form & by dephosphorylation by a phosphatase that causes an increase in activity. 14 5) The kinase is activated by increases in the: • ATP]/[ADP], [acetylCoA]/[CoA], & [NADH]/[NAD+] ratios. • Thus, PDH is inhibited not only by a high-energy potential but also when fatty acids are being oxidized. 1. PDH kinase is, itself, inhibited by ADP & pyruvate. Thus, when activation of glycolysis increases pyruvate levels, PDH kinase is inhibited & PDH remains in an active, nonphosphorylated form. 2. Insulin may increase the amount of PDH complex. 15 • Arsenic Poisoning: it is caused by the presence of a large number of different Arsenious compounds that are effective metabolic inhibitors. Acute accidental or intentional arsenic poisoning requires 2- 2- high doses and involves Arsenate (AsO4 ) & arsenite (AsO ). 16 • Arsenite, is 10 times more toxic than arsenate, it binds to neighboring –SH groups, such as those in dihydrolipoate & in cysteine pairs (vicinal) found in - keto acid dehydrogenase complexes and in succinic dehydrogenase. • Arsenate weakly inhibits enzymatic reactions involving phosphate, including the enzyme glyceraldehydes 3-P dehydrogenase in glycolysis (see Chapter 2). • Thus both aerobic and anaerobic ATP production can be inhibited. The low doses of arsenic compounds found in water supplies are a major public health concern, but are associated with increased risk of cancer rather than direct toxicity. 17 CITRIC ACID CYCLE • The Citric Acid Cycle (CAC) also called Tricarboxylic acid cycle (TCA cycle), or the Krebs cycle plays several roles in metabolism. It is the final pathway where the oxidative metabolism of carbohydrates, amino acids & fatty acids converge, their carbon skeletons being converted to CO2 and H2O. 18 • The cycle occurs in the mitochondria in close proximity to the electron transport, to oxidize the reduced coenzymes produced by the cycle. • The TCA cycle is an aerobic pathway, because O2 is required as the final electron acceptor. • The CAC also participates in a number of important synthetic reactions e.g., heme. Some intermediates of TCA cycle can synthesized by some amino acids. 19 The Overall Pattern of the Citric Acid Cycle: 20 The CAC has 8 Steps that can be divided into TWO Phases: •- First Phase: Removal of 2 mole of CO2 (reaction 1 to 5). •- The 2nd Phase: Regeneration of Oxaloacetate (reaction 6 to 8). 21 Phase One: Removal of Two Molecules of CO2: • Reaction1: Citrate synthase (CS) condenses Oxaloacetate (OAA) with AcetylCoA to form Citrate, by an irreversible aldol condensation to generate a highly unstable Citroyl-CoA which hydrolyzes to yield Citrate. 22 • No Coenzymes Required • The reaction is highly exergonic & has been considered to be a site of regulation for the overall pathway. • Citrate synthase can react with monofluoroacetyl-CoA to form a result of fluoroacetate poisoning. Monofluorocitrate is a potent inhibitor of the aconitase reaction. Monofluorocitrate results in a nearly complete block of CAC activity. 23 • Reaction 2: Isomerization of Citrate to Isocitrate via cis-Aconitate Citrate contains 3ry alcoholic group, which is difficult to oxidize. Aconitase generates Isocitrate the 2ry alcoholic compound, which is more readily oxidized. • The reaction involves successive dehydration & hydration, through cis aconitate as a dehydrated intermediate. 24 • Aconitase does not require cofactors, but requires Fe2+ that may be involved in an iron sulfur center, which is an essential in hydration & dehydration activity of aconitase. • Aconitase is highly stereospecific, it operates on that part of citrate derived from oxaloacetate. Therefore; the carbon atoms lost during the first turn of the cycle orginated in oxaloacetate, not acetyl CoA. 25 • Reaction 3: 1st Oxidative Decarboxylation By Isocitrate Dhydrogenase st st • In this step the 1 of 3 NADH & 1 of 2 CO2 are produced by CAC 26 • Isocitrate converted to α-ketogutarate (α-KG) in NAD-dependent oxidative decarboxylation reaction. • ICDH is one of the rate-limiting steps of the CAC cycle. • ICDH required Mn2+ or Mg2+ for Decarboxylation of β-C of Oxalosuccinate. • ICDH is an Isozyme. In mammalian the extramitochondrial form uses NADP+ whereas the intramitochondrial form uses NAD+; however heart mitochondria contain both forms. • ADP is a +ve effectors but ATP & NADH are -ve allosteric effectors. So, at ATP/ADP & NADH/NAD+ ratios, ICDH is inhibited. 27 • Reaction 4: Generation nd of the 2 CO2 by Oxidative Decarboxylation of α- Ketoglutarate (α-Keto- acid) & Formation of Succinyl-CoA: 28 • α-KGDH is multi-enzyme identical to PDH complex, whears α-Keto acid substrate undergoes oxidative decarboxylation, with formation of an acyl-CoA. • They are also similar in their structural features, TPP, lipoic acid, CoASH, FAD & NAD+ participate in the catalytic mechanism. • α-KGDH consists of α-KG dehydrogenase (E1), dihydrolipoy1 trans- succinylase (E2) & dihydrolipoyl dehydrogenase (E3). nd nd • The 2 CO2 & the 2 NADH of the CAC are produced by α-KGDH. • . Unlike PDH, the α-KGDH is not regulated by a protein kinase. • ATP, GTP, NADH & succinyl CoA inhibit α-KGDH. • Ca2+ activate α-KGDH. • Note: the 2 carbon atoms lost as 2 CO2 are not the same as the 2 atoms introduced as acetyl-CoA. 29 • Reaction 5: A Substrate- level Phosphorylation by Succinate Thiokinase (succinyl-CoA synthetase): 30 • Succinly CoA is an energy-rich thiolester similar to acetyl CoA. & its energy is used to drive the formation of an energy-rich phosphate bond GTP at substrate level. GTP drives the synthesis of ATP via nucleoside diphosphokinase: • GTP + ADP →→ GDP + ATP 31 Phase Two: Regeneration of Oxaloacetate • Reaction 6: Oxidation of Succinate to Fumarate by Succinate Dehyrogenase: 32 • FAD is more powerful oxidant than NAD, FAD is used as coenzyme for SDH, because C-C bond is more difficult to oxidize than a C-O bond. • In conjunction with Fe-S center in SDH, it is tightly bound to the mitochondrial inner membrane unlike other CAC enzymes, which are in matrix. This because FADH2, must be reodixidized with the mitochondrial electron transport system, this is to permit the enzyme to act again. • SDH is activated by Succinate. SDH strongly inhibited by Malonate & OAA. There is very close structural