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 similarly between Malonate & Succinate.

33 Reaction 7: Hydration of Fumarate to Malate by Fumarase • Fumarase reaction is freely reversible but strongly stereospecific. • Funarase catalyzes hydration of trans double bond of Fumarate but not the cis of Maleate. In reverse direction, Fumarase is stereospecific to L- Malate. D-Malate is Not a Substrate.

34 Reaction 8: Oxidation of Malate to Oxaloacetate By Malate Dehydrogenase

35 • Finally, CAC is completed with the NAD+ dependent dehydrogenation of Malate to Oxaloacetate by Malate Dehydrogenase (MDH). • Six isozymes of MDH have been found in human tissues. MDH & LDH are increasesd in presence of tissue damage in certain diseases. Since the isozymes of LDH are easier to assay than those of MDH, the clinical assay of the latter is not routine. • OAA is regenerated for another round of the cycle. The reaction proceeds to the right because the highly exergonic CS reaction keeps intramitochondrial OAA levels low due to the rapid utilization of OAA by CS, in addition NADH produced in the cycle is oxidized rapidly to NAD in the mitochondrial respiratory chain. 36 Stoichiometry of the Citric Acid Cycle:

• The net reaction of the citric acid cycle is

• Two carbon atoms of acetyl unit leave the cycle in the form of 2 CO2 in the successive decarboxylations catalyzed by ICDH & α-KGDH. • Four pairs of hydrogen atoms leave the cycle in 4 oxidation reactions. Three NAD+ molecules are reduced in the oxidative decarboxylations of isocitrate and α-Ketoglutarate & oxidation of malate. 37 • One FAD molecule reduced in the formation of Fumarate from Succinate • One ATP generated from energy-rich thioester linkage in succinyl CoA.

• Two H2O molecules consumed in by the hydrolysis of citroyl CoA, & in hydration of fumarate. • Three ATP are formed for each NADH in the mitochondrion. Two ATP are generated per FADH2.

38 Reactions of the CAC

39 • Molecular oxygen does not participate directly in CAC. The cycle operates only under aerobic condition due to regeneration of NAD & FAD in the mitochondrion occurs only by the transfer of electrons to molecular oxygen. • The following equation for catabolsim of glucose through glycolysis and CAC cycle:

• Most of the ATP generated from the re-oxidation of reduced electron carries in the respiratory chain

40 Regulation of the Citric Acid Cycle

• CAC adjusted to meet the cell’s needs for ATP & other Biosynthetics Intermediates. • First, AcetylCoA, is a crucial factor in determining the rate of the Krebs cycle. • Second, regulatory influences on PDH have an important effect on CAC activity. • Third, The interrupt in the supply of oxygen, the continuous supply of ADP or the source of NADH &/or FADH2 would shut down cycle activity.

41 A) Regulation of PDH:

• Note: the PHD complex is not a part of the Krebs cycle, but its regulation enhances the sensitivity with which the Krebs cycle is controlled. • Two states of the cell are important in the regulation of PDH by covalent modification. First, PHD responds to the energy of the cell. When the ATP concentration is high, glycolysis slows, & the activity of the PHD is lowered. • Second, PHD is sensitive to the oxidation reduction state of the cell. Any alteration of NAD+, NADH, NADP+ & NADPH, the PHD will be affected.

42 B) Control & Inhibitors of CAC:

• 1) Citrate Synthase is considered as an important control point. • CS is stimulated by the availability of its 2 substrates, Acetyl CoA & oxaloacetate. • Competitive feedback inhibition: NADH inhibits CS & Succinyl-CoA competes with acetyl-CoA in CS reaction

43 2) ICDH, α-KGDH & to a lesser extent, MDH is key sites for allosteric regulation of the CAC. • NAD+ is a substrate for these 3 enzymes, as well as for PDH, therefore the (NAD+)/(NADH) ratio is the most important factor controlling CAC activity. • The NAD+-linked ICDH is stimulated by ADP & AMP and is inhibited by ATP and NADH. • Hence, under high-energy conditions (high ATP/ADP, Pi & high NADH/NAD+ratios) the NAD-ICDH of CAC is inhibited. • During periods of low energy, the activity of this enzyme is stimulated in order to accelerate energy generation in the CAC. • -KGDH activity is inhibited by Succinyl-CoA & by NADH.

44 Regulation of Citric Acid Cycle

45 The Citric Acid Cycle Interacts with Other Metabolic Pathways • CAC is Amphibolic because it acts as Catabolic & Anabolic. The CAC also serves as a cross road for the inter conversion of various metabolites & as an important source of biosynthetic intermediates, such as • 1- Succinyl CoA is used in the synthesis of Heme & other Porphyrins.

46 • 2- Citrate can be transported from the mitochondria to the cytosol, where it cleaved to provide Acetyl CoA for biosynthesis of fatty acid & sterol. • 3- Oxaloacetate & α-ketoglutarate (keto acids), have carbon that are homologous to aspartate & glutamate. Pyruvate is homologous to alanine. • When these amino acids exceed the requirements for protein biosynthesis, the excess can readily be converted to krebs cycle intermediates to produce energy. The reversible interconversion of these α-keto acids are catalyzed by Transaminases (aminotransferases).

47 Efflux of intermediates from the TCA cycle

48 • In the liver, TCA cycle intermediates are continuously withdrawn into the pathways of fatty acid synthesis, amino acid synthesis, gluconeogenesis, and heme synthesis. • In brain, -ketoglutarate is converted to glutamate and GABA, both neurotransmitters. Since the krebs cycle intermediates interacts with other metabolic pathways, these reactions tend to deplete CAC intermediates & the cycle would be impaired. • Therefore, the existence of other processes that replenish the stores of CAC intermediates must be present to prevent the impairment of the cycle. These reactions are called Anaplerotic pathways means "filling up".

49 Anaplerotic Reactions

• Major Anaplerotic Pathways: (C3 C4) interconverstions. • 1. Transaminase reactions: conversion of Alanine, Aspartate & Glutamate into Pyruvate, Oxaloacetate, & α- Ketoglutarate, respectively. These reactions are the first steps in gluconeogenesis or lipogenesis from amino acids. • 2. Glutamate dehydrogenase, presents another route for synthesis of α-ketoglutarate from glutamate. + • Glutamate + NAD(P) +H2O------α-ketoglutarate + NAD (P)H + + NH4

50 3. The Malic enzyme: generation of Pyruvate & acetylCoA from C4 CAC-intermediates in mitochondria, production of NADPH in cell cytoplasm from amino acids for lipid biosynthesis & release of CO2 in plants. This enzyme is required for lipogenesis (fatty acid synthesis) from most amino acids

51 4. Formation of PEP by PEP Carboxykinase (PEPCK) • Animal PEPCK found in mitochondrion (liver) and cytoplasm (heart & skeletal muscle) for gluconeogenesis.

52 5. Formation of Oxaloacetate In mammals

• the main anaplerotic enzyme is ATP dependent Pyruvate Carboxylase (PC), it's the first in gluconeogenesis. • Acetyl CoA is required as activator to promote gluconeogenesis in Liver & Kidney. PC Widely distributed in Euokaryots & Prokaryots

53 6. Formation of Oxaloacetate in higher Plants, Yeast, Bacteria • PEP Carboxylase does not require ATP.

54 7. Excess Citrate can be exported out of the mitochondria • into the cytosol; where it is broken down by ATP-citrate lyase to yield OAA & acetyl-CoA. Acetyl-CoA produced is a precursor for fatty acids synthesis. • OAA is reduced to Malate or OAA can be oxidatively decarboxylated to Pyruvate by malic enzyme. The formed Malate & Pyruvate can be transported back into the mitochondrial matrix to replenish the CAC.

55 The Glyoxylate Cycle

• Notice that Isocitrate lyase & Malate synthase are unique to the glyoxylate cycle & 2 acetyl groups enter the cycle and four carbons leave as succinate. • The reactions catalyzed by Isocitrate Lyase & Malate Synthase bypass the 5 CAC steps between isocitrate & malate. • Glyoxylate cycle bypass the two decarboxylation steps of the CAC makes the net formation of succinate, oxaloacetate & other cycle intermediates from acetyl-CoA. • The 2 carbons of acetyl-CoA can be incorporated into oxaloacetate, which is an efficient gluconeogenic • The succinate generated is transported from the glyoxysome to the mitochondrion, Where it is converted to OAA., which utilized for glucose synthesis via gluconeogenesis.

56 The Glyoxylate Cycle

57 • The Glyoxylate cycle is active in the germinating seeds of some plants & in certain m.o. that can live on acetate as the sole carbon source & to carry out the net synthesis of carbohydrate from fat. Plants synthesize sugars by using the glyoxylate cycle, which can be considered an anabolic variant from of the CAC. • In plants, Glyoxylate cycle occurs in glyoxysomes, a specialized organ that carries out both β-oxidation of FAs to acetyl-CoA & utilization of acetyl-CoA in the cycle.

58 • The glyoxylate cycle allows many m.o. to metabolize two-carbon of acetate. Escherichia coli can grow in a medium that provides acetate as the sole carbon source, as can many fungi, protozoa's, and algae. • Vertebrates lack the Glyoxylate cycle & cannot synthesize glucose from acetate or the fatty acids that give rise to acetyl-CoA., because the PDH is irreversible. Net reaction of the Glyoxylate cycle is:

• 2 Acetyl CoA + NAD+ + 2H2O →→ Succinate + 2 CoA + NADH + 2 H+

59 THANK YOU

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