生物化学生化课件chapter

Chapter19GlycolysisGlycolysisisthesequenceofreactionsthatmetabolizesonemoleculeofglucosetotwomoleculesofpyruvate丙酮酸withtheitantnetproductionoftwomoleculesofATP.Anoverviewofkeystructuresandreactions

thestructureofthereactantsHexose己糖ortriose丙糖andtheirderivativesphosphorylated

Ester酯oranhydride酸酐thekindsofreactionsPhosphoryltransfer转移bykinase激酶Phosphorylshift移位bymutase变位酶Isomerizationbyisomerase异构酶Dehydrationbydehydratase脱水酶Aldolcleavage醛裂byaldolase醛缩酶compartment:thecellcytosolStage1of

GlycolysisFormationofF-1,6-BPfromGlc

Thestrategyoftheseinitialstepsistotrapthesubstrateinthecellandformacompoundthatcanbereadilycleavedintophosphorylatedthree-carbonunits.

将葡萄糖(从胞外)截获到细胞内,形成一个可容易地切割(成两个磷酸化三碳单位)的化合物(F-1,6-BP)Thefirststageconsistsof3steps:Glc→G-6-P,byHK己糖激酶,phosphorylationG-6-P→F-6-P,byPGlcisomerase磷酸葡萄糖异构酶,isomerizationF-6-P→F-1,6-BP,byPFK磷酸果糖激酶,phosphorylationStage2of

glycolysisFormationofglyceraldehyde3-phosphate3-磷酸甘油醛bycleavage醛裂andisomerizationThesecondstageconsistsof2steps

(图示为3+2+5模式;老版本3+4+3模式)II(1)F-1,6-BP→GAl3-P+DHAP磷酸二羟丙酮,by

aldolase醛缩酶,

aldolcleavage

II(2)DHAP→GAl3-P,by

TIM

磷酸丙糖异构酶,isomerizationStage3of

glycolysisEnergyconservation:phosphorylationiscoupledtotheoxidationofGAl-3-PⅢ(1)GAl-3-P→1,3-BPG1,3-二磷酸甘油酸,byGAl-3-Pdehydrogenase脱氢酶

phosphorylationiscoupledtotheoxidationofGAl-3-P磷酸化与氧化相偶联phosphorylationcoupledtooxidation1,3-BPGisahigh-potentialphosphorylcompoundFormationofATPfrom1,3-BPGⅢ(2)

1,3-BPG→3-PGA,by

phosphoglyceratekinase,PGAkinase磷酸甘油酸激酶Thefirstenergy-generatingreactioninEMP(Embden-Meyerhof-Parnaspathway)

Ⅲ(1)+Ⅲ(2):

aldehyde→carboxylicacid

NAD+→NADHADP→ATPFormationofPyrandthegenerationofasecondATPThefollowing3steps:3-PGA→Pyr3-PGA→2-PGA,byPGAmutase磷酸甘油酸变位酶,phosphorylshift2-PGA→PEP磷酸烯醇化丙酮酸,byenolase烯醇化酶,dehydration脱水PEP→Pyr,byPyrkinase,phosphoryltransferTherearrangementisthepreludetothenextdehydration坝重排是脱水的前提PEP(justlike1,3-BPG)hasahighphosphoryltransferpotentialthesecondATPformed.Stagesofglycolysis

(1)Glcistrappedanddestabilized(2)twointerconvertiblethree-carbonmoleculesaregeneratedbycleavageofsix-carbonfructose(3)ATPisgenerated3+2+5模式`EnergyyieldintheconversionofGlcintoPyrCarbonatoms:asix-carbonGlc→2three-carbonPyrHydrogenatomsandNAD+

2NADH(潜在能量)formedEnergy:2ATP(直接能量)formedEntryofFruandGal半乳糖intoglycolysisFru→F-1-PDHAP+GAl→GAl-3-PGal→Gal-1-P+UDP-Glc

G-1-P+UDP-Gal→UDP-Glc

G-1-PG-6-PGalishighlytoxicifthetransferaseismissingThecauseofgalactosemia半乳糖血症:absenceofGal-1-Puridyltransferase半乳糖1-磷酸尿苷酸转移酶Thetypeofinheritence:autosomalrecessive常染色体隐性

Symptoms:vomitingordiarhea,enlargementoftheliverandjaundice,mentalretardationCauseofDamage:byanaccumulationoftoxicsubstances—galactitol半乳糖醇(reducedfromGal)转移酶缺陷→1-磷酸半乳糖无法转移到尿苷上→半乳糖代谢受阻→(积累的)半乳糖被还原成半乳糖醇→致病Diagnosticcriterion:absenceofthetransferaseinredcellsTreatment:exclusionofGalfromthediet

PFKisthekeyenzymeinthecontrolofglycolysis

Thedualroleofglycolysis:togenerateATPandtoprovidebuildingblocks酵解具双重功能:产生能量和构建元件TherateofconversionofGlcintoPyrisdecidedby:activitiesofenzymes(catalyzingtheirreversiblereactions)regulatedbythereversiblebindingofallostericeffectors(suchasATP/AMP,H+,citrate,F-2,6-BP)orbycovalentmodification酶的活性amountsofthesekeyenzymes,variedbytranscriptionalcontrol

酶的数量Inmetabolicpathways,enzymescatalyzingessentiallyirreversiblereactionsarepotentialsitesofcontrol(HK,PFK,PK).在代谢途径中,催化基本不可逆反应的酶是潜在的调控位点.PFKisthemostimportantcontrolelementintheglycolyticpathwayofmammals.Itisinhibitedby:highlevelofATP(reversedbyAMP)能量H+(topreventsexcessiveformationoflactateandaprecipitousdropinbloodpH)低pH值(酸性)Citrate柠檬酸(asasignalofbuildingblock),byenhancingtheinhibitoryeffectofATP构建元件F-2,6-BPisanallostericactivator变构激活剂(byincreasingPFK’saffinityforF-6-PanddiminishingtheinhibitoryeffectofATP)AregulatedbifunctionalenzymesynthesizesanddegradesF-2,6-BPF-6-P→F-2,6-BP,byPFK2

磷酸果糖激酶2(F-6-P→F-1,6-BP,byPFK);thereversalreaction:F-2,6-BP→F-6-P,byFBPase2果糖二磷酸酶2(F-1,6-BP→F-6-P,byFBPase)

bothPFK2andFBPase2arepresentinasinglepolypeptidechain.

Thisbifunctionalenzymecontains:N-terminalregulatorydomainkinasedomain激酶区phosphatasedomain.磷酸酶区Itispossiblyarisedbythefusionofgenesencodingthekinaseandphosphatasedomains.此双功能酶可能由原本分立的两个基因(分别编码激酶和磷酸酶)融合而成feedforwardstimulation前馈促进F-6-P→F-2,6-BP→PFK→F-1,6-BPSubstrate→effector→enzyme→product(substratestimulatestheformationofproduct)TheactivitiesofPFK2andFBPase2arereciprocallycontrolledbyphosphorylation

ofasingleserineresidue.Glc↓→glucagon胰高血糖素inblood↑→cAMPcascade→itsphosphorylation→FBPase2↑andPFK2↓→F-2,6-BP↓→glycolysis↓HKandPKalsosetthepaceofglycolysis

TheinhibitionofPFKleadstotheinhibitionofHK

PFKinactive→F-6-P↑F-6-P↑→G-6-P↑HKinhibited(1:HK;3:PFK;10:PK)Thereisglucokinase(GK)葡萄糖激酶inliver

itisaspecializedisoformofHK

notinhibitedbyG-6-PGKphosphorylatesGlconlywhenGlcisabundantbecaseithasaveryhighKmforGlcthandoesHK.

theroleofGKistoprovideG-6-Pforthesynthesisofglycogen

thehighKmofGKinthelivergivesbrainandmusclefirstcallonGlcwhenGlclimited

当血液中[Glc]很高时Glc可不受限制地(不受G-6-P抑制)摄入(到肝细胞);血液中Glc水平低下时,肝细胞不与急需Glc的脑和肌肉细胞争夺Glc(GK的Km值很高),充分说明了肝脏的利他性.WhyisPFKthepacemakerofglycolysis?(notHK)

G-6-Pisnotsolelyaglycolyticintermediate(e.g.aspentosephosphatepathwayintermediate)它既作为糖酵解,又作为磷酸戊糖途径的中间物thefirstirreversiblereactionuniquetoapathwayiscalledcommittedstep(限速步骤)theenzymecatalyzingthecommittedstepinametabolicsequenceisthemostimportantcontrolelementinthepathwayPKisregulatedbymanyfactors(alsocontrolledbyreversiblephosphorylation)itsactivator:F-2,6-BP;

itsinhibits:ATP(whenEChigh);Ala(asabuildingblocksignal)

alsocontrolledbyreversiblephosphorylation:bloodGlc↓→glucagon→cAMPcascade级联放大系统→phosphorylationofPK→itsactivity↓hormonesignal(e.g.vasopressin加压素)→[Ca2+]↑→phosphorylationofPK→itsactivity↓底物前体,产物对等物,能荷,共价修饰,激素信号Thesehormone-triggeredphosphorylations,likethatofthebifunctionalenzymecontrollingthelevelsofF-2,6-BP,preventtheliverfromconsumingGlcwhenitismoreurgentlyneededbybrainandmuscle.DiversefatesofPyr:ethanol乙醇,lactate乳酸,oracetylCoA

thefateofPyrisvariable

Ethanol(Pyr→acetaldehyde乙醛→Ethanol),inyeastandseveralothermicroorganismsLactateinavarietyofmicroorganisms(orinhigherorganismswhentheamountofO2islimited)TheregenerationofNAD+inthereductionofPyrtolactateorethanolsustainsthecontinuedoperationofglycolysisunderanaerobic（厌氧）conditions.丙酮酸(走回头路)还原使NAD+再生,这维持了在厌(缺)氧条件下酵解的持续进行Pyr→acetylCoA(byPyrDHcomplex)→CO2(byTCAcycle),aerobically（有氧）ThebindingsiteforNAD+isverysimilarinmanyDHs（脱氢酶）

TheNAD+-bindingregioncommontoLacDH,alcoholDH,GAl-3-PDHandmalateDHisafundamentalstructuralmotifofNAD+-linkedDHs.上述脱氢酶与辅酶(NAD+-)的结合区是相似的madeupof4αhelicesandasheetof6parallelβstrandsTheadeninemoietyofNAD+inahydrophobiccrevice疏水裂隙thereactivesideofthenicotinamideunit(ofLacDHandalcoholDH)inapolarenvironment(coenzymestereospecificitytypeB)thereactivesideofthenicotinamideunit(ofGAl-3-PDHandmalateDH)inapolarenvironment(coenzymestereospecificitytypeA)上述脱氢酶的辅酶都是NAD+,而且(所有脱氢酶之间)有相似的(酶与辅酶)结合模式,腺苷酸部分都在疏水裂隙,尼克酰胺环的活性位点则在极性区;按尼克酰胺环的排布又分成A和B型(环平面旋转1800).Inducedfit诱导契合inHK:Glcclosestheactive-sitecleft活性部位裂隙

Substrate底物Glcinducesthemovementoftheenzyme(HK)backboneTheclosingofthecleftinHKisastrikingexampleoftheroleofinducedfitinenzymeactionSubstrate-inducedcleftclosingislikelytobeageneralfeatureofkinase.底物(Glc)与酶(HK)结合后诱导酶骨架运动,使得酶的裂隙关闭(只露出底物的6号碳原子,以接受由ATP转移来的磷酸基).Aldolase醛缩酶formsaSchiffbase希夫碱withDHAPThecondensationprocessofDHAPandGAl-3-PtoformF-1,6-BP(thereverseoftheglycolyticreaction)质子化希夫碱(作为电子穴)形成→促进烯醇化阴离子形成→与带正电的甘油醛缩合DHAPformsaprotonated质子化SchiffbasewithaspecificLysresidueDHAP+酶中特定的Lys残基中的ε氨基

TheresultingprotonatedSchiffbaseplaysacriticalroleincatalysis.Itpromotestheformationoftheenolateanion烯醇化阴离子ofDHAPbyservingaselectronsink,apotentelectronacceptor.electronsink电子穴:

electrophilic亲电子atomorgroupofatomsthatcancaptureanelectronfromanotherpartofamolecularsystem.enolateanion+aldehydegroupofGAl-3-P→protonatedSchiffbaseofF-1,6-BPitsdeprotonationandhydrolyzation去质子化和水解Kineticperfectionincatalysis:triose丙糖phosphateisomerase(TIM)inactionTIMcatalyzesanintramolecularoxidation-reductiontransferofahydrogenatomfromC-1toC-2DHAPintoGAl-3-P(aketose酮糖intoanaldose醛糖)throughanenediol烯二醇intermediateTIMisanexampleofakineticallyperfectenzymeAthioester硫酯isformedintheoxidationofGAl-3-PGAl-3-P→1,3-BPG,anoxidativephosphorylationreactionCatalyticmechanismofGAl-3-PDHadditionofanucleophile亲核物质(-SHgroupintheenzyme)thecarbonatominaldehydesubstratelesspositivelychargedhydrideion氢阴离子readilyleavestheadditioncompoundhydrideiontransferredtoNAD+additionofPiandanacylphosphateformedThioesterisanenergy-richintermediate,whichcouplesan‘unfavorable’reaction(fromacarboxylatetoanacylphosphate)witha‘favorable’one(theoxidationofanaldehyde).Arsenate砷酸(AsA,AsO43－),ananalogofphosphate,poisonsbyuncouplingoxidationandphosphorylationarsenateuncouplesoxidationandphosphorylationglycolysisproceedsbutnoATPformedinthisstepbyformingahighlylabileacylarsenateArseni