3Catabolism of proteins, fats, and carbohydrates in the three stages of cellular respiration Stage 1: oxidation of fatty acids, glucose, and some amino acids yields acetyl CoA.Stage 2: oxidation of acetyl groups in the citric acid cycle includes four steps in which electrons are abstractedStage 3: Electrons carried by NADH and FADH2 are funneled into a chain of mitochondrial electron carriers--- the respiratory chain--- reducing to O2 to H2O--- production of ATP
6Overall reaction catalyzed by the pyruvate dehydrogenase complex (PDH), and coenzyme A A oxidative decarboxylation —an irreversible oxidation (3 enzyme complexes; 5 coenzymes).The –SH group of the mercapto-ethylamine moiety forms a thioester with acetate in acetyl-coenzymes A.similar to a-ketoglutarate dehydrogenase, of the citric acid cycle, and the branched-chain a-keto acid dehydrogenase, of the oxidative pathways of several amino acidsthioesters high acyl group transfer potential
8Fates of Acetyl CoA In the presence of CHO an using energy Metabolized to CO2, NADH, FADH2,GTP and, ultimately, ATPIf energy not being used (Lots of ATP present)Made into fatIf energy being used, but no CHO presentStarvationForms ketone bodies (see fat metabolism slides)Danger!
9Recurring Motifs in Metabolism “Activated” electron and functional group carriers
10The Pyruvate Dehydrogenase Complex Requires Five Coenzymes five different coenzymes require for dehydrogenation and decarboxylation of pyruvate to the acetyl: (1). thiamine pyrophosphate (TPP), (2). flavin adenine dinucleotide (FAD), (3). coenzyme A (CoA-SH, (4). nicotinamide adenine dinucleotide (NAD), and (5). lipoate.lipoate has two thiol groups: reversible oxidation to a disulfide bond (-S-S-): electron hydrogen carrier and acyl carrier.
11In Substrate Channeling, Intermediates Never Leave the Enzyme Surface pyruvate reacts with the bound thiamine pyrophosphate (TPP) of (E1) -decarboxylation to the hydroxyethyl derivativeE1 transfer two electrons and the acetyl group from TPP to the oxidized form of the lipoyllysyl group of E2, - form acetyl thioester-SH group of CoA replaces the -SH group of E2 - acetyl-CoA and the fully reduced dithiolE3 transfer of two hydrogen atoms from the reduced lipoyl groups of E2 to the FAD prosthetic group of E3, restoring the oxidized form of the lipoyllysyl group of E2.the reduced FADH2 of E3 transfers a hydride ion to NAD, forming NADH.substrate channeling: All enzymes and coenzymes are clustered, allowing the intermediates to react quickly without diffusing away from the surface of the enzyme complex.
12Pyruvate dehydrogenase Complex (PDH) It is a multi-enzyme complex containing three enzymes associated together non-covalently:E-1 : Pyruvate dehydrogenase , uses Thiamine pyrophosphate as cofactor bound to E1E-2 : Dihydrolipoyl transacetylase, Lipoic acid bound, CoA as substrateE-3 : Dihydrolipoyl Dehydrogenase FAD bound, NAD+ as substrateAdvantages of multienzyme complex:Higher rate of reaction: Because product of one enzyme acts as a substrate of other, and is available for the active site of next enzyme without much diffusion.Minimum side reaction.Coordinated control.
13metabolonsmetabolons = multienzyme complexes ensure efficient passage of the product of one enzyme reaction to the next enzyme in the pathway.Certain enzymes of the citric acid cycle have been isolated together as supra molecular aggregates, or have been found associated with the inner mitochondrial membrane, or have been shown to diffuse in the mitochondrial matrix more slowly than expected for the individual protein in solution - Substrate Channeling
14Biological tethersThe cofactors lipoate, biotin, and the combination of mercaptoethylamine and pantothenate form long, flexible arms in the enzymes to which they are covalently bound, acting as tethers that move intermediates from one active site to the next.
15Arsenic Compound poisoning: Inactivation of E-2 of PDC, and other proteins. Organic Arsenical were used as antibiotics for the treatment of syphilis and trypanosomiasis.Micro-organisms are more sensitive to organic arsenicals than humans.But these compounds had severe side effects and As-poisoning.Fowler’s solution, the famous 19th century tonic contained 10mg/ml As. Charles Darwin died of As poisoning by taking this tonic.Napoleon Bonaparte’s death was also suspected to be due to As poisoning.
17Historical perspective: 1930: Elucidation of GlycolysisStudy of oxidation of glucose in muscle, addition of Malonate inhibited the respiration (i.e. O2 uptake).Malonate is an inhibitor of Succinate oxidation to Fumerate1935: Szent-Gyorgyi: demonstrated that little amounts (catalytic amounts) of succinate, fumerate, malate or oxaloacetate acelerated the rate of respiration.He also showed the sequence of inter-conversion:Succinate --- Fumerate --- malate ---oxaloacetate.1936: Martius & Knoop: Found the following sequence of reaction:Citrate to cis-aconitase to Isocitrate to a Ketogluterate to succinate1937: Krebs: Enzymatic conversion of Pyruvate + Oxaloacetate to citrate and CO2Discovered the cycle of these reactions and found it to be a major pathway for pyruvate oxidation in muscle.
18Overall goal Makes ATP Makes NADH Makes FADH2 Requires some carbohydrate to run
19Geography Glycolysis in the cytosol Krebs in mitochondrial matrix MitochondrionOuter membrane very permeableSpace between membranes called intermembrane space (clever huh!)Inner membrane (cristae)Permeable to pyruvate,Impermeable to fatty acids, NAD, etcMatrix is inside inner membrane
20Names of enzyme Are Confusing! Synthases (No ATP, GTP…)A+BABSynthetases (needs ATP, GTP) = LigaseLyaseABA+BKinase (ATP)P+AAATPPhosphorylaseAPAB+Pi+BPhosphatasePAA+PiSynthases catalyze condensation reactions in which no nucleoside triphosphate (ATP, GTP, and so forth) is required as an energy source Synthetases catalyze condensations that do use ATP or another nucleoside triphosphate as a source of energy for the synthetic reaction. Ligases are enzymes that catalyze condensation reactions in which two atoms are joined using ATP or another energy source. (Thus synthetases are ligases.)kinase is applied to enzymes that transfer a phosphoryl group from a nucleoside triphosphate such as ATP to an acceptor moleculephosphorylase catalyse a displacement reaction in which it use phosphate (Pi) attacks substrate which result in breaking the substrate and Pi becomes covalently attached at the point of bond breakagePhosphatase: the removal of a phosphoryl group from a phosphate ester
22Tricarboxylic acid (TCA) or Krebs cycle FluoroacetateArsenateMalonate
23open form1Formation of citrateclose formCondensation of acetyl-CoA with oxaloacetate to form citrate.Methyl carbon of acetyl group is joined of the carbonyl group of oxaloacetate (OAA)The flexible domain binds OAA - induce large conformational change and create a binding site for acetyl-CoA- another conformation change to release CoA.
24Mechanisms for enzyme-catalyzed bisubstrate reactions P. 208The enzyme and both substrates come together to form aternary complex. In ordered binding, substrate 1 must bindbefore substrate 2 can bind productively.(b) An enzyme-substrate complex forms, a product leaves thecomplex, the altered enzyme forms a second complex withanother substrate molecule, and the second product leaves,regenerating the enzyme.
25Formation of isocitrate via cis-aconitate 2Formation of isocitrate via cis-aconitateAconitase catalyzes the reversible transformation of citrate to isocitrate , the intermediary formation of the cis-aconitate--- does not dissociated from the active site– Aconitase can promote the reversible addition of H2O to the double bond of enzyme-bound cis-aconitate in two ways: citrate and isocitrateAconitase contains an ironsulfur center which acts both in the binding of the substrate at the active site and in the catalytic addition or removal of H2O.
26Oxidation of isocitrate to a-ketoglutarate and CO2 3Oxidation of isocitrate to a-ketoglutarate and CO2Decarboxylation of isocitrate to a-ketoglutarate by isocitrate dehydrogenase.Two forms of isocitrate dehydrogenase (NAD +---in mitochondrial matrix; NADP +---both mitochondrial matrix and cytosol). isocitrate, loses one carbon by oxidative decarboxylation.isocitrate binds to the enzyme and is oxidized by hydride transfer to NAD+ or NADP+,Interaction of the carbonyl oxygen with a bound Mn2+ ion increases the electron-withdrawing capacity of the carbonyl group and facilitates the decarboxylation step.by rearrangement of the enol intermediate to generate a -ketoglutarate.
27Oxidation of a-ketoglutarate to succinyl-CoA and CO2 4Oxidation of a-ketoglutarate to succinyl-CoA and CO2Oxidative decarboxylation by a-ketoglutarate DHaseThe reaction is Identical to the pyruvate DHase reaction--- both enzymes are similar structurally and functionally --- common evolutionary origin.
28Conversion of Succinyl-CoA to succinate 5Conversion of Succinyl-CoA to succinateSuccinyl-CoA similar to acetyl-CoA has strongly negative standard free energy of hydrolysis (Succinyl-CoA synthase or succinic thiokinase).A substrate –level phosphorylation (GTP to ATP by nucleoside diphosphate kinase)
29Oxidation of succinate to fumarate 6Succinate is oxidized to fumarate (succinate DHase--- membrane-bound).Malonate, and analog of succinate– is a strong competitive inhibitor of succinate DHase and therefore block the activity of TCA cycle.The Enzyme also contain iron-sulfur clusters which bind both the substrate and FAD.1 FADH2 = 1.5 ATP
30Hydration of fumarate to malate 7Hydration of fumarate to malate(Trans-)(Trans-)(Cis-)L-Fumarase is highly stereospecific; it catalyzes hydration of the trans double bond of fumarate but not the cis double bond of maleate (the cis isomer of fumarate). Inthe reverse direction (from L-malate to fumarate), fumarase is equally stereospecific: D-malate is not a substrate.
31Oxidation of Malate to oxaloacetate 8Oxidation of Malate to oxaloacetateIn intact cells oxaloacetate is continually removed by the highly exergonic (-32.2 KJ/mol) citrate synthase reactionThis keeps oxaloacetate concentration in the cell extremely low (<10-6 M)--- toward right
32Products of one turn of the citric acid cycle Three NADH, one FADH2 and one GTP(ATP), and two CO2 are release in oxidative decarboxylation reaction. (are not the same two carbons that entered in the form of the acetyl group; additional turn)Most of the reactions are reversible (except 3 reactions)Two e- from NADH to O2 (2.5 ATP) ; Two e- from FADH2 to O2 (1.5 ATP)Start from acetoyl-CoA: 3 X X = 10 ATP/cycle/Acetyl-CoAglucose CO2 and H2O => ATP(65 %). ref. to Table 16-112311
33The energy of oxidations in the cycle is efficiently conserved
35Glycerol 3-phosphate shuttle (Skeletal muscle and brain) Skeletal muscle and brain use a different NADH shuttle, the glycerol 3-phosphate shuttle It differs from the malate-aspartate shuttle in that it delivers the reducing equivalents from NADH to ubiquinone and thus into Complex III, not Complex I providing only enough energy to synthesize 1.5 ATP molecules per pair of electrons.
36Biosynthetic precursors produced by an incomplete citric acid cycle in anaerobic bacteria Early anaerobes most probably used some of the reactions of the citric acid cycle in linear biosynthetic processes.some modern anaerobic microorganisms use an incomplete citric acid cycle as a source of, not energy, but biosynthetic precursorsthe first three reactions of the cycle to make - ketoglutarate but, lacking a-ketoglutarate dehydrogenase, they cannot carry out the complete set of citric acid cycle reactions.four enzymes that catalyze the reversible conversion of oxaloacetate to succinyl-CoA and can produce malate, fumarate, succinate, and succinyl-CoA from oxaloacetate in a reversal reaction.
37Role of the citric acid cycle in anabolism the citric acid cycle is an amphibolic pathway, one that serves in both catabolic and anabolic processes.Besides its role in the oxidative catabolism of carbohydrates, fatty acids, and amino acids, the cycle provides precursors for many biosynthetic pathwaysa –Ketoglutarate and oxaloacetate can, serve as precursors of the amino acids aspartate and glutamate by simple transaminationThrough aspartate and glutamate, the carbons of oxaloacetate and a-ketoglutarate are then used to build other amino acids, as well as purine and pyrimidine nucleotides.Oxaloacetate is converted to glucose in gluconeogenesis .Succinyl- CoA is a central intermediate in the synthesis of the porphyrin ring of heme groups, which serve as oxygen carriers (in hemoglobin and myoglobin) and electron carriers (in cytochromes)
38Anaplerotic reactions anaplerotic reactions: replenish the intermediates of the citric acid cycle that are removed to serve as biosynthetic precursors => concentrations of the citric acid cycle intermediates remain almost constant
40Citrate: A Symmetrical Molecule That Reacts Asymmetrically BOX 16-2Citrate: A Symmetrical Molecule That Reacts Asymmetricallythe labeled a-ketoglutarate isolated from the tissue suspension contained 14C only in the g-carboxyl group.
42Enzymatic reaction of prochiral molecules BOX 16-2Enzymatic reaction of prochiral moleculesabgthe active site of aconitase may have three points to which the citrate must be bound and that the citrate must undergo a specific three-point attachment to these binding points. The binding of citrate to three such points could happen in only one way, and this would account for the formation of only one type of labeled a-ketoglutarate.prochiral molecules: Organic molecules, such as citrate, that have no chiral center but are potentially capable of reacting asymmetrically with an asymmetric active site
4316.3 Regulations of Citric Acid Cycle 16.4 The Glyoxylate Cycle
44Regulation of metabolite flow from pyruvate through the citric acid cycle Pyruvate DHase kinaseThe pyruvate DHase complex is regulated by allosteric and covalent mechanisms.Three factors govern the rate of flux through the cycle:(1) substrate availability,(2) inhibition by accumulating products, and(3) allosteric feedback inhibition of the enzymes.
45REGULATIONThe pyruvate DHase is inhibited by covalent protein modification through reversible phosphorylation of a specific Ser on one of the two subunits of E1 (by Pyruvate DHase kinase which is activate by ATP) => inactivate E1
47Glyoxylate cycle TCA cycle Vertebrates cannot convert fatty acids or acetate to carbohydratesConversion of PEP to pyruvate and pyruvate to acetyl-CoA are irreversible.In plants and certain invertebrates, some microorganisms use glyoxylate cycleIsocitrate lyase and malate synthase are not in TCAGlyoxysome (not all time) is in lipid-rich seeds during germination. Also contains all enzymes need for degradation of the fatty acids stored in seed oils.TCA cycle
48The Citric Acid and Glyoxylate Cycles Are Coordinately Regulated Glyoxysome (not all time) is in lipid-rich seeds during germination. Also contains all enzymes need for degradation of the fatty acids stored in seed oils.The reactions of the glyoxylate cycle (in glyoxysomes) proceed simultaneously and mesh with, TCA (mitochondria), as intermediates pass between these compartments.
49Regulation of isocitrate dehydrogenase activity that determines partitioning of isocitrate between the glyoxylate and citric acid cyclesA bifunctional proteinFor distinct pathways: fatty acid to acetyl-CoA, glyoxylate cycle (glyoxysome), citric acid cycle (mitochondria); gluconeogenesis (cytosol)Isocitate DHase (protein Kinase) Phosphoryled - inactivated; --to glyoxylate cycle---glucoseProtein phosphatase— remove P---activate isocitate DHase--- TCA---energy.Phosphatase activate isocitrate DHase --- stimulated by intermediates of TCA and glycolysis and by reduced cellular energy; same factor inhibits the protein kinaseThe same intermediates of the glycolytic and citric acid cycles inhibits isocitrate lyase--- When energy-yielding metabolism is sufficiently fast to keep the concentration of glycolytic and TCA intermediates low, isocitrate deHase inactivated, the inhibition is removed, and isocitrate flows into glycolyte.