13 Biochemistry _ Glycolysis

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  • 1. Lecture 13 Glycolysis

2. CatabolismCatabolism is the set of metabolicpathways that breaks down moleculesinto smaller units to release energy 3. Central Role of GlucoseMajor pathways of glucose utilization 4. Glycolysis Carried out by nearly every living cell In cytosol of eukaryotes Catabolic process Releases energy stored in covalent bonds Stepwise degradation Glucose Other simple sugars 5. Overview of glycolysis 6. Two phases of glycolysisGlucose + 6O2 = 6CO2 + 6H2O DGo= -2840 kJ/molGlucose + 2NAD+ = 2Pyruvate + 2NADH + 2H+ DGo = -146 kJ/mol5.2% of total free energy that can be released by glucose is released inglycolysis. 7. Reaction 1: phosphorylationPhosphorylation of glucoseGlycolysis takes place in the cytosol of cells.Glucose enters the Glycolysis pathway by conversion to glucose-6-phosphate.Initially there is energy input corresponding to cleavage of two ~P bonds of ATP.Kinase: An enzyme that catalyzes the phosphorylation of a molecule using ATP. 8. Glycolysis - Enzyme mechanismsPictorial analogy: water represents flux of metabolites, amount of water in flaskrepresents amount of a particular intermediate, pipes between flasks are enzymes, verticaldrop represents decrease in free energyDG = height difference between flask bottoms (The change in standard Gibbs free energy,the formation of 1 mole substance)DG = height difference between water levels (The change in Gibbs free energy )Koelle, lec15, p21 9. Hexokinase catalyzes:Glucose + ATP glucose-6-P + ADPThe reaction involves nucleophilic attack of the C6hydroxyl O of glucose on P of the terminal phosphateof ATP.ATP binds to the enzyme as a complex with Mg++.NNNH2N NOCH2H HHOH OHHOO O-O P O P O P OO- O-O-adenineriboseATPadenosine triphosphate 10. Induced fit:Glucose bindingto Hexokinasestabilizes aconformationin which: the C6 hydroxyl of thebound glucose is close tothe terminal phosphate ofATP, promoting catalysis. water is excluded from the active site.This prevents the enzyme from catalyzing ATPhydrolysis, rather than transfer of phosphate to glucose. 11. Hexokinase reactionI. This enzyme is present in most cells. In liver Glucokinaseis the main hexokinase which prefers glucose assubstrate.II. It requires Mg-ATP complex as substrate. Un-complexedATP is a potent competitive inhibitor of this enzyme.III. Enzyme catalyses the reaction by proximity effect;bringing the two substrate in close proximity.IV. This enzyme undergoes large conformational change uponbinding with Glucose. It is inhibited allosterically by G6P. 12. Reaction 2: isomerizationConversion of glucose 6-phosphate to fructose 6-phosphatealdose ketoseIsomerase: An enzyme that catalyzes the transformation of compounds intotheir positional isomers. In the case of sugars this usually involves theinterconversion of an aldose into a ketose, or vice versa. 13. Phosphoglucose Isomerase catalyzes:glucose-6-P (aldose) fructose-6-P (ketose)The mechanism involves acid/base catalysis.The acid/base catalysis, and is thought to proceed via an enediol intermediate, as withPhosphoglucose Isomerase.Active site Glu and His residues are thought to extract and donate protons during catalysis. 14. Reaction 3: phosphorylation 15. CH2OPO32-Phosphofructokinase61 CH2OPO3CH2OH2OH5HOH HO4 3OH H62-CH2OPO32OH2-5HOH HO4 3OH H1ATP ADPMg2+fructose-6-phosphate fructose-1,6-bisphosphatePhosphofructokinase catalyzes:fructose-6-P + ATP fructose-1,6-bisP + ADPThis highly spontaneous reaction has a mechanism similarto that of Hexokinase.The Phosphofructokinase reaction is the rate-limiting stepof Glycolysis.The enzyme is highly regulated. 16. Phosphofructokinase-1 Reaction: Transfer ofphosphoryl group from ATP to C-1 ofF6P to produce Fructose 1,6 bisphosphate.I. This step is an important irreversible, regulatory step.II. The enzyme Phosphofructokinase-1 is one of the most complexregulatory enzymes, with various allosteric inhibitors andactivators.III. ATP is an allosteric inhibitor, and Fructose 2,6 bisphosphateis an activator of this enzyme.IV. ADP and AMP also activate PFK-1 whereas citrate is aninhibitor. 17. Reaction 4: cleavage 18. 1CH2OPO32HO 3HH OHH OHCCCCCH2OPO34562-Aldolase2-O321CH2OPO32-COCH2OHH OCCCH2OPO3123+ H OHfructose-1,6-bisphosphatedihydroxyacetone glyceraldehyde-3-phosphate phosphateTriosephosphate IsomeraseAldolase catalyzes: fructose-1,6-bisphosphate 2-dihydroxyacetone-P + glyceraldehyde-3-PThe reaction is an aldol cleavage, the reverse of an aldol condensation.Note that C atoms are renumbered in products of Aldolase. 19. Schiff base intermediate ofAldolase reactionlysineA lysine residue at the active site functions in catalysis.The keto group of fructose-1,6-bisphosphate reacts with the e-amino group of the active site lysine, to form a protonatedintermediate.Cleavage of the bond between C3 & C4 follows. 20. Reaction 5: isomerizationTriose Phosphate Isomerase (TIM) catalyzes:dihydroxyacetone-P glyceraldehyde-3-P 21. Triose Phosphate Isomerase (TPI)Triose phosphate isomerase (TPI)-> Isomerisation accelerated 1010-fold-> Kcat/Km = 2*108 M-1 s-1 -> kineticallyperfect enzyme-> suppresses an undesired side reactionReaction 100 times fasterTPI traps enediol intermediate -> prevents sidereaction -> opens again when GAP formed 22. Reaction 6: oxidationGlyceraldehyde-3-phosphate Dehydrogenase catalyzes:glyceraldehyde-3-P + NAD+ + Pi 1,3-bisphosphoglycerate + NADH + H+ 23. Covalent catalysis 24. Reaction 7: substrate level phosphorylationPhosphoglycerate Kinase catalyzes:1,3-bisphosphoglycerate + ADP 3-phosphoglycerate + ATPThis phosphate transfer is reversible. 25. Reaction 8: shift of phosphoryl groupPhosphoglycerate Mutase catalyzes:3-phosphoglycerate 2-phosphoglycerateMutase: An enzyme that catalyzes the transposition of functional groups,such as phosphates, sulfates, etc. 26. An active site histidineside-chain participates inPi transfer, by donating &accepting phosphate.The process involves a2,3-bisphosphateintermediate.CCCH2OPO32-O O-1H OPO32-232,3-bisphosphoglycerate 27. Phosphoglycerate Mutase Reaction: Conversion of 3-phosphoglycerate to 2-phosphoglycerate (2-PG).I. In active form, the phosphoglycerate mutase is phosphorylatedat His-179.II. There is transfer of the phosphoryl group from enzyme to 3-PG,generating enzyme bound 2,3-biphosphoglycerate intermediate.This compound has been observed occasionally in reactionmixture.III. In the last step of reaction the phosphoryl group from the C-3 ofthe intermediate is transferred to the enzyme and 2-PG isreleased.IV. In most cells 2,3BPG is present in trace amount, but inerythrocytes it is present in significant amount. There it regulatesoxygen affinity to hemoglobin. 28. Reaction 9: dehydrationEnolase catalyzes:2-phosphoglycerate phosphoenolpyruvate + H2OThis dehydration reaction is Mg++-dependent. 29. Reaction 10: substrate level phosphorylationPyruvate Kinase catalyzes:phosphoenolpyruvate + ADP pyruvate + ATP 30. glucose GlycolysisATPADPglucose-6-phosphateHexokinasePhosphoglucose Isomerasefructose-6-phosphateATPADPfructose-1,6-bisphosphatePhosphofructokinaseAldolaseglyceraldehyde-3-phosphate + dihydroxyacetone-phosphateTriosephosphateIsomeraseGlycolysis continued 31. glyceraldehyde-3-phosphateNAD+ + PiNADH + H+Glyceraldehyde-3-phosphateDehydrogenase1,3-bisphosphoglycerateADPATP3-phosphoglyceratePhosphoglycerate KinasePhosphoglycerate Mutase2-phosphoglycerateH2OEnolasephosphoenolpyruvateADPATPPyruvate KinasepyruvateGlycolysiscontinued. 32. There are 10 enzyme-catalyzed reactions in glycolysis.There are two stagesStage 1: (Reactions 1-5) A preparatory stage in which glucose is phosphorylated,converted to fructose which is again phosphorylated and cleaved into twomolecules of glyceraldehyde-3-phosphate. In this phase there is an investment oftwo molecules of ATP.Stage 2: (Reactions 6-10) The two molecules of glyceraldehyde-3-phosphate areconverted to pyruvate with the generation of four ATP molecules and twomolecules of NADH. Thus there is a net gain of two ATP molecules per molecule ofGlucose in glycolysis.Importance of phosphorylated intermediates:1. Possession of negative charge which inhibit their diffusion through membrane.2. Conservation of free energy in high energy phosphate bond.3. Facilitation of catalysis. 33. Summary of glycolysis-> 10 reaction steps-> 1 x C-6 (glucose) converted into 2x C-3 (pyruvate)-> oxidation of glucose -> 2 NADH generated-> 2 ATPs used + 4 ATPs generated -> pay off: 2 ATPs33 34. Glycolysis - total pathway, omitting H+:glucose + 2 NAD+ + 2 ADP + 2 Pi 2 pyruvate + 2 NADH + 2 ATPIn aerobic organisms: pyruvate produced in Glycolysis is oxidized to CO2via Krebs Cycle (citric acid cycle) NADH produced in Glycolysis & Krebs Cycle isreoxidized via the respiratory chain, with productionof much additional ATP. 35. Regulation of Glycolysis:Two types controls for metabolic reactions:a) Substrate limited : When concentrations of reactant and productsin the cell are near equilibrium, then it is the availability ofsubstrate which decides the rate of reaction.b) Enzyme-limited: When concentration of substrate and products arefar away from the equilibrium, then it is activity of enzyme thatdecides the rate of reaction.Three steps in glycolysis have enzymes which regulate the flux ofglycolysis.I. The hexokinase (HK)II. The phoshofructokinase (PFK)III. The pyruvate kinase 36. The Glycolysis pathway is regulated by control of 3enzymes:Hexokinase, Phosphofructokinase & Pyruvate Kinase. Local control of metabolism involves regulatory effectsof varied concentrations of pathway substrates orintermediates, to benefit the cell. Global control is for the benefit of the whole organism,& often involves hormone-activated signal cascades.Liver cells have major roles in metabolism, includingmaintaining blood levels various of nutrients such asglucose. Thus global control