- 1.Biochemistry of MetabolismGlycolysisCopyright 1998-2007 by Joyce J. Diwan.All rights reserved.
2. 6 CH OPO 22 3 5OH H H4H1 OHOHOH3 2 H OH glucose-6-phosphateGlycolysis takes place in the cytosol of cells.Glucose enters the Glycolysis pathway by conversionto glucose-6-phosphate.Initially there is energy input corresponding tocleavage of two ~P bonds of ATP. 3. 6 CH2OH6 CH OPO 22 3ATP ADP 5 O 5 O H H HH H H 4 14H1 OHH OHMg2+ OHOHOH OH 3 23 2 H OHHexokinase HOH glucose glucose-6-phosphate1. 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++. 4. NH2 ATPN N adenosine triphosphateOOO NN OPO PO PO CH2adenineOO O O HH HH OH OH riboseMg++ interacts with negatively charged phosphateoxygen atoms, providing charge compensation &promoting a favorable conformation of ATP at theactive site of the Hexokinase enzyme. 5. 6 CH2OH6 CH OPO 22 3ATP ADP 5 O 5 O H H HH H H 4 14H1 OHH OHMg2+ OHOHOH OH 3 23 2 H OHHexokinase HOH glucose glucose-6-phosphateThe reaction catalyzed by Hexokinase is highlyspontaneous.A phosphoanhydride bond of ATP (~P) is cleaved.The phosphate ester formed in glucose-6-phosphatehas a lower DG of hydrolysis. 6. 6 CH2OH 6 CH OPO 2 2 3ATP ADP 5 O5 OInduced fit: H H H H HH 4 1 4H1 OHH 2+ OHGlucose bindingOHOHMgOH OH 2to Hexokinase3 2 3 H OHHexokinase HOHstabilizes a glucose glucose-6-phosphateconformationin which:glucose the C6 hydroxyl of thebound glucose is close tothe terminal phosphate of HexokinaseATP, promoting catalysis. water is excluded from the active site.This prevents the enzyme from catalyzing ATPhydrolysis, rather than transfer of phosphate to glucose. 7. glucose HexokinaseIt is a common motif for an enzyme active site to belocated at an interface between protein domains that areconnected by a flexible hinge region.The structural flexibility allows access to the active site,while permitting precise positioning of active siteresidues, and in some cases exclusion of water, assubstrate binding promotes a particular conformation. 8. 6 CH OPO 22 3 5 6 CH OPO 21CH2OHHOH2 3O H4H15 H HO2 OHOHOH H4 3 OH3 2 OHH H OHPhosphoglucose Isomeraseglucose-6-phosphate fructose-6-phosphate2. Phosphoglucose Isomerase catalyzes: glucose-6-P (aldose) fructose-6-P (ketose)The mechanism involves acid/base catalysis, with ringopening, isomerization via an enediolate intermediate,and then ring closure. A similar reaction catalyzed byTriosephosphate Isomerase will be presented in detail. 9. Phosphofructokinase 6 CH OPO 21CH2OH6 CH OPO 21CH2OPO32 2 32 3 O ATP ADP O5 HHO25 H HO2H 43 OHMg2+ H43 OHOHH OH Hfructose-6-phosphatefructose-1,6-bisphosphate3. Phosphofructokinase 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, as will be discussed later. 10. 21CH2OPO32CO HOHO 3C HAldolase 3CH2OPO321C H 4C OH2C O+ H 2C OH 2H C OH1CH2OH3 CH2OPO356 CH2OPO32dihydroxyacetone glyceraldehyde-3-phosphate phosphatefructose-1,6-bisphosphateTriosephosphate Isomerase4. Aldolase catalyzes: fructose-1,6-bisphosphate dihydroxyacetone-P + glyceraldehyde-3-PThe reaction is an aldol cleavage, the reverse of an aldolcondensation.Note that C atoms are renumbered in products of Aldolase. 11. lysine1CH2OPO32 H+2C NH (CH2)4 Enzyme H 3NC COO + HO 3CH CH2H 4COH CH2H 5COH CH26 CH2OPO32 CH2Schiff base intermediate of NH3Aldolase reactionA lysine residue at the active site functions in catalysis.The keto group of fructose-1,6-bisphosphate reacts withthe e-amino group of the active site lysine, to form aprotonated Schiff base intermediate.Cleavage of the bond between C3 & C4 follows. 12. 2 1CH2OPO3 2COHOHO 3CHAldolase 3 CH2OPO321C H 4COH2C O+ H 2C OH2 H C OH1CH2OH3 CH2OPO3 5 6 CH2OPO32dihydroxyacetone glyceraldehyde-3- phosphate phosphate fructose-1,6- bisphosphate Triosephosphate Isomerase5. Triose Phosphate Isomerase (TIM) catalyzes: dihydroxyacetone-P glyceraldehyde-3-PGlycolysis continues from glyceraldehyde-3-P. TIMs Keqfavors dihydroxyacetone-P. Removal of glyceraldehyde-3-Pby a subsequent spontaneous reaction allows throughput. 13. Triosephosphate IsomeraseH H OHH+O+ + +H C OH H HCH HCC O C OHH C OHCH2OPO32 CH2OPO32 CH2OPO32dihydroxyacetoneenediol glyceraldehyde- phosphateintermediate3-phosphateThe ketose/aldose conversion involves acid/base catalysis,and is thought to proceed via an enediol intermediate, aswith Phosphoglucose Isomerase.Active site Glu and His residues are thought to extract anddonate protons during catalysis. 14. OH HC O O OCCCH2OPO32CH2OPO32 proposed phosphoglycolateenediolate transition state intermediateanalog2-Phosphoglycolate is a transition state analog thatbinds tightly at the active site of Triose PhosphateIsomerase (TIM).This inhibitor of catalysis by TIM is similar in structure tothe proposed enediolate intermediate.TIM is judged a "perfect enzyme." Reaction rate is limitedonly by the rate that substrate collides with the enzyme. 15. Triosephosphate Isomerasestructure is an ab barrel, orTIM barrel.In an ab barrel there are8 parallel b-strands surroundedby 8 a-helices.Short loops connect alternating TIMb-strands & a-helices. 16. TIM barrels serve as scaffoldsfor active site residues in adiverse array of enzymes.Residues of the active site arealways at the same end of thebarrel, on C-terminal ends ofb-strands & loops connecting TIMthese to a-helices.There is debate whether the many different enzymes withTIM barrel structures are evolutionarily related.In spite of the structural similarities there is tremendousdiversity in catalytic functions of these enzymes andlittle sequence homology. 17. OHHC O O O CC CH2OPO32CH2OPO32proposed phosphoglycolate enediolate transition stateTIM intermediateanalogExplore the structure of the Triosephosphate Isomerase(TIM) homodimer, with the transition state inhibitor2-phosphoglycolate bound to one of the TIM monomers.Note the structure of the TIM barrel, and the loop thatforms a lid that closes over the active site after bindingof the substrate. 18. Glyceraldehyde-3-phosphateDehydrogenase H O + H+ OOPO32 1C NAD+ NADH 1C + PiHC OHH C OH 2 2 3 CH2OPO32 3 CH2OPO32glyceraldehyde-1,3-bisphospho- 3-phosphate glycerate6. Glyceraldehyde-3-phosphate Dehydrogenasecatalyzes: glyceraldehyde-3-P + NAD+ + Pi 1,3-bisphosphoglycerate + NADH + H+ 19. Glyceraldehyde-3-phosphate DehydrogenaseH O + H+ OOPO321C NAD+ NADH 1C+ Pi HC OHH C OH2 23 CH2OPO32 3 CH2OPO32 glyceraldehyde-1,3-bisphospho-3-phosphate glycerateExergonic oxidation of the aldehyde in glyceraldehyde-3-phosphate, to a carboxylic acid, drives formation of anacyl phosphate, a "high energy" bond (~P).This is the only step in Glycolysis in which NAD+ isreduced to NADH. 20. H OH1C H3N+ C COO H 2 C OHCH2 3 CH2OPO32SHglyceraldehyde-3- cysteine phosphateA cysteine thiol at the active site of Glyceraldehyde-3-phosphate Dehydrogenase has a role in catalysis.The aldehyde of glyceraldehyde-3-phosphate reactswith the cysteine thiol to form a thiohemiacetalintermediate. 21. Enz-Cys SH O OH HC CHCH2OPO32 glyceraldehyde-3- OH OH phosphateOxidation to a Enz-Cys S CH CH CH2OPO32carboxylic acid NAD+ thiohemiacetal NADHintermediate(in a ~ thioester) OOHoccurs, as NAD+ Enz-Cys S CCH CH2OPO32is reduced toacyl-thioesterPiNADH.intermediate OOH 2 Enz-CysSH O3POCCHCH2OPO321,3-bisphosphoglycerateThe high energy acyl thioester is attacked by Pi toyield the acyl phosphate (~P) product. 22. H O OH HC CNH2 NH2+N + N2e + HR R NAD+ NADHRecall that NAD+ accepts 2 e plus one H+ (a hydride)in going to its reduced form. 23. Phosphoglycerate Kinase OOPO32 ADP ATP OO 1C1CH 2C OH H 2C OHMg2+3 CH2OPO32 3 CH2OPO32 1,3-bisphospho- 3-phosphoglycerateglycerate7. Phosphoglycerate Kinase catalyzes: 1,3-bisphosphoglycerate + ADP 3-phosphoglycerate + ATPThis phosphate transfer is reversible (low DG), sinceone ~P bond is cleaved & another synthesized.The enzyme undergoes substrate-induced conformationalchange similar to that of Hexokinase. 24. Phosphoglycerate Mutase OO O O C 1 C 1H 2C OH H 2C OPO3223 CH2OPO3 3 CH2OH3-phosphoglycerate2-phosphoglycerate8. Phosphoglycerate Mutase catalyzes: 3-phosphoglycerate 2-phosphoglyceratePhosphate is shifted from the OH on C3 to theOH on C2. 25. Phosphoglycerate MutaseO O histidine OOH 1C 1 CH3N+CCOOH 2C OHH 2COPO322CH23 CH2OPO33 CH2OHC3-phosphoglycerate 2-phosphoglycerateHN CH HC NH An active site histidine side-chainparticipates in Pi transfer, bydonating & accepting phosphate.OO 1CThe process involves aH 2C OPO322,3-bisphosphate intermediate. 23 CH2OPO32,3-bisphosphoglycerateView an animation of thePhosphoglycerate Mutase reaction. 26. EnolaseO H OOH OOOOCCC11 H 2 C OPO32C OPO32 2COPO32 3 CH2OH CH2OH3 CH22-phosphoglycerate enolate intermediate phosphoenolpyruvate9. Enolase catalyzes:2-phosphoglycerate phosphoenolpyruvate + H2OThis dehydration reaction is Mg++-dependent.2 Mg++ ions interact with oxygen atoms of the substratecarboxyl group at the active site.The Mg++ ions help to stabilize the enolate anionintermediate that forms when a Lys extracts H+ from C #2. 27. Pyruvate KinaseOO OOADP ATP1 C1 C2 C OPO32 2 C O3 CH2 3 CH3phosphoenolpyruvate pyruvate10. Pyruvate Kinase catalyzes:phosphoenolpyruvate + ADP pyruvate + ATP 28. Pyruvate Kinase OO O OOOC ADP ATPCC 1 1 1 2C OPO32 C 2 OH2C O 3 CH2 3 CH2 3 CH3 phosphoenolpyruvateenolpyruvate pyruvateThis phosphate transfer from PEP to ADP is spontaneous. PEP has a larger DG of phosphate hydrolysis than ATP. Removal of Pi from PEP yields an unstable enol, whichspontaneously converts to the keto form of pyruvate.Required inorganic cations K+ and Mg++ bind to anionicresidues at the active site of Pyruvate Kinase. 29. glucoseGlycolysis ATP Hexokinase ADPglucose-6-phosphate Phosphoglucose Isomerasefructose-6-phosphate ATPPhosphofr