生物化學-考研課件-ch14-酶動力學-enzyme-kinetics

BiochemistryForSpecialityofBiotechnologyorBioengineeringatCSUMr.XIAJinlanBiochemistryForSpecialityof1Chapter14EnzymekineticsIntroductiontoenzymes

酶（學）導論Factorsaffectingenzymeactivity

影響酶活性的因素EnzymeassaysEnzymekineticsandinhibition

酶動力學和抑制作用Chapter14EnzymekineticsIn2Introductiontoenzymes

酶（學）導論Thefeaturesofenzymes酶的特征Sometermsofenzyme酶的幾種術語Classesofenzymes酶的種類EnzymeNomenclature酶的命名法Traditionalnomenclature傳統(tǒng)命名法Systematicnomenclature系統(tǒng)命名法Expressionofenzymeactivity酶活性的表示Componentsofenzyme酶的成分Cofactors輔助因子Introductiontoenzymes

酶（學）導3PowerfulCatalyticpower強的催化力Increasereactionrateupto1016foldMakereactiontakenplaceunderrelativelymildconditionsoftemperatureandpHHighspecificity高的特異性TothesubstratesthattheyactonandtheproductstheyformEnzymeactivitycanberegulated酶活性可以被調控Basicallyareproteins,exceptfewribozymes除了少量的核酶外,基本上是蛋白質Enzymeshavefourbasicfeatures

酶的四個基本特征PowerfulCatalyticpower強的催化力4Sometermsofenzymes酶的幾種術語Proteoenzyme蛋白酶Ribozyme核酶Abzyme抗體酶Holoenzyme全酶Apoenzyme脫輔酶Multimericenzyme多聚合體酶(多亞基酶)Sometermsofenzymes酶的幾種術語Pr5Enzymesareclassifiedintosixclassesbasedonthetypeofreactioncatalyzed

基于催化反應的類型,酶可分為六種類型Oxidoreductases氧化還原酶Transferofelectrons電子的轉移Transferases轉化(或轉移)酶Transferoffunctionalgroups分子間功能基團的轉移Hydrolases水解酶Hydrolysisreactions水解反應Lyases裂解酶CleavageofC-C,C-O,C-Netc.,oftenformingadoublebondC-C,C-O,C-N鍵的裂解,并通常形成雙鍵的反應Isomerases異構酶Transferofgroupswithinamolecule分子內基團的轉移Ligases(orsynthases)鏈接酶(或合成酶)BondformationcoupledtoATPhydrolysis伴隨ATP水解的鍵合Enzymesareclassifiedintosi6EnzymesareclassifiedintosixclassesbasedonthetypeofreactioncatalyzedNameTypeofreactioncatalyzed

ExampleOxido-reductasesTransferofelectronsA-+BA+B-AlcoholdehydrogenaseTransfer-asesTransferoffunctionalgroupsA-B+CA+B-CHexokinaseHydrolasesHydrolysisreactionsA-B+H2OA-H+B-OHTrypsinLyasesCleavageofC-C,C-O,C-Netc.,oftenformingadoublebondPyruvatedecarboxylaseIsomerasesTransferofgroupswithinamoleculeMaleateisomaseLigases(orsynthases)BondformationcoupledtoATPhydrolysisPyruvatecarboxylaseA+BA-BX-A-B-YY-A-B-XX-A-B-YA=B+Y-XEnzymesareclassifiedintosi7TraditionalNomenclatureofenzymesTraditionalNomenclatureofen8Systematicnomenclature:

Eachenzymeisuniquelyidentifiedbya four-digitclassificationnumber

Enzymecommission(EC)number:Four-digitclassificationnumberWhatdothesenumberssignify?TrypsinEC3.4.21.4ChymotrypsinEC3.4.21.1ElastaseEC3.4.21.36Systematicnomenclature:

E9Expressionofenzymeactivity

酶活性的表示

Initialrate(V0)ofthereaction

mmolofsubstratetransformedperminute每分鐘轉化底物的微摩爾數V0:Xmmol.min-1

Enzymeunit酶的單位,SIunitTheamountthatcatalyzestheformationofonemmolofproductperminute每分鐘催化1mmol產物的形成所需的酶量U:mmol.min-1Katal,SIunitAmountofenzymecatalyzingtheconversionofonemoleofsubstratetoproductpersecond每秒鐘催化轉化1摩爾底物所需的酶量Katal:mol.s-1Expressionofenzymeactivity10Expressionofenzymeactivity酶活性的表示

11Expressionofenzymeactivity酶11Someexpressionsofthecomponentsofanenzyme酶的成分的幾種表示方式Enzyme酶Proteinpart+Cofactors(Non-proteinpart)(蛋白部分+輔助因子(非蛋白部分))Holoenzyme全酶Apoenzyme+Prostheticgroup(脫輔基酶+輔基)Someexpressionsofthecompon12Cofactors輔助因子InorganicInorganicion(s)

無機離子Fe2+,Zn2+,Cu2+,etc.Inorganiccluster(s)無機簇Fe-Scluster,Mn-Ocomplex,etcOrganicCoenzyme輔酶Whichlooselybindtotheirenzymes

Prostheticgroup輔基WhichtightlybindtotheirenzymesCofactors輔助因子Inorganic13Theimportantcoenzymesandprostheticgroups重要的輔酶和輔基Coenzymes輔酶CoenzymeA(CoA)NAD(H),NADP(H)Nicotinamideadeninedinucleotide煙酰胺腺嘌呤二核苷酸NicotinamideadeninedinucleotidephosphateProstheticgroups輔基FAD(H2),FMN(H2)*Flavinadeninedinucleotide黃素腺嘌呤二核苷酸Flavinmononucleotide黃素單核苷酸Biotin生物素Heme血紅素*FADandFMNinmostcasesareprostheticgroupsbindingtoflavinproteinsTheimportantcoenzymesandpr14Watersolublevitamins(VitaminB)Thiamine(B1)硫胺素InCoenzymes：thiaminepyrophosphate存在于硫胺焦磷酸輔酶中Riboflavin(核黃素B2)Asprostheticgroupflavinprotein:FADandFMNNicotinicacid(niacin煙堿酸)InCoenzymes:NADandNADPPantothenicacid(泛酸）InCoenzymeABiotin(生物素）Inprostheticgroup:Biocytin生物胞素Lipidsolublevitamins(VitaminA,D,E,K)VitaminARetinal視網膜素：vision,growthandreproductionMostcoenzymesarederivedfromvitamins

大多數輔酶源于維生素Watersolublevitamins(Vitami15Somecommoncoenzymes,theirvitaminprecursorsanddeficiencydiseasesCoenzyme/prostheticgroupPrecursorDeficiencydiseaseCoenzymeAPanthothenicacidDermatitis皮膚炎FAD,FMNRiboflavin(VitaminB2)GrowthretardationNAD+

(CoenzymeI),NADP+

(CoenzymeII)Niacin(菸堿酸)Pellagra玉蜀黍疹ThiaminepyrophosphateThiamine(VitaminB1)Beriberi腳氣病TetrahydrofolateFolicacidAnemia貧血癥Deoxyadenosylcobalamin（脫氧腺苷鈷胺素）Cobalamin(VitaminB12)Perniciousanemia惡性貧血癥Co-substrateinthehydroxylationofprolineincollagenVitaminC(ascorbicacid)Scurvy壞血病Pyridoxalphosphate磷酸吡哆醛Pyridoxine(vitaminB6)Dermatitis皮膚炎Somecommoncoenzymes,theirv16CoenzymeATransferofacetylgroupCoenzymeATransferofacetylg17NAD+,NADP+CoenzymeofDehydrogenasesNAD+,NADP+Coenzymeof18NAD(P)++H++2e-NAD(P)HReductionOxdationNAD+,NADP+H++2e-H-NAD(P)++H-NAD(P)HReductionOxdationGeneralreactionNAD(P)++H++2e-NAD(P)HRe19FAD,FMNFig.18.21,p591Isoalloxazine異咯嗪Flavinmononucleotide,FMNFlavinadeninedinucleotide,FADRiboflavinAMPRibitol核醣醇FAD,FMNFig.18.21,p591Isoall20FAD,FMNFAD+2H++2e-FADH2ReductionOxdationFMN+2H++2e-FMNH2ReductionOxdationFAD,FMNFAD+2H++2e-FADH21ChemicalstructureofbiotinChemicalstructureofbiotin22Chapter14EnzymekineticsIntroductiontoenzymes

酶（學）導論Factorsaffectingenzymeactivity

影響酶活性的因素EnzymeassaysEnzymekineticsandinhibition

酶動力學和抑制作用Chapter14EnzymekineticsIn23Cofactors輔助因子(SEEthelastsection)

pHTemperatureSubstrateconcentration底物濃度ProductconcentrationFactorsaffectingenzymeactivity

Chapter14EnzymesCofactors輔助因子(SEEthelasts24EffectofpH

Pepsin胃蛋白酶Papain木瓜蛋白酶Trypsin胰蛋白酶Cholinesterase膽堿酯酶過氧化氫酶EffectofpHPepsinPapain木瓜蛋白酶T25Effectoftemperature<40°C,increasewithriseoftemperature>50°C,decreaseduetodenaturationofenzymeBATypicallyEffectoftemperature<40°C,in26Enzymeassays酶的檢測TwomostconvenientwaysDetg.therateofappearanceofproduct測定產物形成的速率Detg.therateofdisappearanceofsubstrate測定反應物減少的速率Alinkedenzymeassayway酶聯檢測法Firstenzymereaction+secondenzymereactionMeasurements測定Spectrophotometry分光光度法Enzymeelectrode酶電極法Enzymeassays酶的檢測Twomostcon27LDH:lactatedehydrogenase乳酸脫氫酶COO-CHOHCH3+NAD+COO-COCH3Pyruvate丙酮酸Lactate乳酸+H+NADH+A340nmLDHLDH:lactatedehydrogenase乳酸28葡萄糖氧化酶過氧化氫氧化酶葡萄糖酸葡萄糖氧化酶過氧化氫氧化酶葡萄糖酸29Chapter14EnzymekineticsIntroductiontoenzymes

酶（學）導論Factorsaffectingenzymeactivity

影響酶活性的因素EnzymeassaysEnzymekineticsandinhibition

酶動力學和抑制作用Chapter14EnzymekineticsIn30Enzymekineticsandinhibition

(酶動力學和抑制作用)Catalyticmechanism催化機制Enzymekinetics酶動力學Michaelis-MentenmodelEnzymeinhibition酶的抑制Reversibleinhibition可逆抑制Nonreversibleinhibition非可逆抑制Enzymekineticsandinhibition31EnzymescatalyzechemicalreactionthroughloweringitsactivationenergyG<0:thermodynamicallyfavorableG>0:notthermodynamicallyfavorableProgressofreactionFreeenergy,GTransitionstate(uncatalyzed)Transitionstate(catalyzed)G?uncatalyzedG?catalyzedSPGEnzymescatalyzechemicalreac32ActivationEnergy~RateConstantofReactionG?~k

Arrheniusequationk=Ae-G?/RTG?kActivationEnergy~RateConst33Questions(selfstudy)

Whatdoesdynamicequilibriummean?Enzymescanacceleratetheattainmentoftheequilibriumposition,canitchangethisposition?What’sroleofactivesiteofanenzyme?Bywhichforcesthesubstratebindstotheactivesiteofanenzyme?Chemicalequilibrium:(selfstudy)Questions(selfstudy)Chemica34Michaelis-MentenmodelHyperboliccurve雙曲線Michaelis-MentenmodelHyperbol35Doublereciprocalorlineweaver-BurkplotDoublereciprocalorlineweave36V0Time(min)Amountofproductformed(mmol)MeasurementandcalculationofV0V0Time(min)Amountofproduct37Enzymeinhibition酶的抑制Inhibition抑制的定義Aphenomenonofloweringenzyme’scatalyticrate.Inhibitor抑制劑AnymoleculewhichactsdirectlyonanenzymetoloweritscatalyticrateTwomaintypesofinhibitionsIrreversible非可逆抑制(抑制)Reversible可逆(抑制)Competitive競爭性~Noncompetitive非競爭性~Mixednoncompetitiveinhibition混合型非競爭性~Enzymeinhibition酶的抑制Inhibiti38IrreversibleinhibitionbycovalentbindingbetweenIandE

ResiduestobeboundSusceptibleaminoacidresiduesneartheactivesiteSer,withOHgroupCys,withSHgroupPrincipleChangemolecularconformationorinhibitapproachingofthesubstratetotheactivesiteofenzymeI:inhibitorE:enzymeS:substrateIrreversibleinhibitionbycov39Diisopropylphospho-fluoridate(DIPF)二異丙基磷酰氟ExamplesDiisopropylphospho-fluoridate40Reversiblecompetitiveinhibition

BothIandScompetewithEattheactivesite活性位點.

Ibinds(noncovalently)onlytoE

I,S:closelysimilarinstructureReversiblecompetitiveinhibit41I,S:closelysimilarinstructureI,S:closelysimilarinstruc42Reversiblenoncompetitiveinhibition

Theinhibitorbindsreversiblyatasiteotherthantheactivesite（非活性位點）IbindsnoncovalentlytobothEandES

Reversiblenoncompetitiveinhi43MixednoncompetitiveinhibitionIbindsatasitedistinctfromactivesite,yetaffectthebindingofSattheactivesiteMixednoncompetitiveinhibitio44HomeworkProblems1-7Pp458-459HomeworkProblems1-745BiochemistryForSpecialityofBiotechnologyorBioengineeringatCSUMr.XIAJinlanBiochemistryForSpecialityof46Chapter14EnzymekineticsIntroductiontoenzymes

酶（學）導論Factorsaffectingenzymeactivity

影響酶活性的因素EnzymeassaysEnzymekineticsandinhibition

酶動力學和抑制作用Chapter14EnzymekineticsIn47Introductiontoenzymes

酶（學）導論Thefeaturesofenzymes酶的特征Sometermsofenzyme酶的幾種術語Classesofenzymes酶的種類EnzymeNomenclature酶的命名法Traditionalnomenclature傳統(tǒng)命名法Systematicnomenclature系統(tǒng)命名法Expressionofenzymeactivity酶活性的表示Componentsofenzyme酶的成分Cofactors輔助因子Introductiontoenzymes

酶（學）導48PowerfulCatalyticpower強的催化力Increasereactionrateupto1016foldMakereactiontakenplaceunderrelativelymildconditionsoftemperatureandpHHighspecificity高的特異性TothesubstratesthattheyactonandtheproductstheyformEnzymeactivitycanberegulated酶活性可以被調控Basicallyareproteins,exceptfewribozymes除了少量的核酶外,基本上是蛋白質Enzymeshavefourbasicfeatures

酶的四個基本特征PowerfulCatalyticpower強的催化力49Sometermsofenzymes酶的幾種術語Proteoenzyme蛋白酶Ribozyme核酶Abzyme抗體酶Holoenzyme全酶Apoenzyme脫輔酶Multimericenzyme多聚合體酶(多亞基酶)Sometermsofenzymes酶的幾種術語Pr50Enzymesareclassifiedintosixclassesbasedonthetypeofreactioncatalyzed

基于催化反應的類型,酶可分為六種類型Oxidoreductases氧化還原酶Transferofelectrons電子的轉移Transferases轉化(或轉移)酶Transferoffunctionalgroups分子間功能基團的轉移Hydrolases水解酶Hydrolysisreactions水解反應Lyases裂解酶CleavageofC-C,C-O,C-Netc.,oftenformingadoublebondC-C,C-O,C-N鍵的裂解,并通常形成雙鍵的反應Isomerases異構酶Transferofgroupswithinamolecule分子內基團的轉移Ligases(orsynthases)鏈接酶(或合成酶)BondformationcoupledtoATPhydrolysis伴隨ATP水解的鍵合Enzymesareclassifiedintosi51EnzymesareclassifiedintosixclassesbasedonthetypeofreactioncatalyzedNameTypeofreactioncatalyzed

ExampleOxido-reductasesTransferofelectronsA-+BA+B-AlcoholdehydrogenaseTransfer-asesTransferoffunctionalgroupsA-B+CA+B-CHexokinaseHydrolasesHydrolysisreactionsA-B+H2OA-H+B-OHTrypsinLyasesCleavageofC-C,C-O,C-Netc.,oftenformingadoublebondPyruvatedecarboxylaseIsomerasesTransferofgroupswithinamoleculeMaleateisomaseLigases(orsynthases)BondformationcoupledtoATPhydrolysisPyruvatecarboxylaseA+BA-BX-A-B-YY-A-B-XX-A-B-YA=B+Y-XEnzymesareclassifiedintosi52TraditionalNomenclatureofenzymesTraditionalNomenclatureofen53Systematicnomenclature:

Eachenzymeisuniquelyidentifiedbya four-digitclassificationnumber

Enzymecommission(EC)number:Four-digitclassificationnumberWhatdothesenumberssignify?TrypsinEC3.4.21.4ChymotrypsinEC3.4.21.1ElastaseEC3.4.21.36Systematicnomenclature:

E54Expressionofenzymeactivity

酶活性的表示

Initialrate(V0)ofthereaction

mmolofsubstratetransformedperminute每分鐘轉化底物的微摩爾數V0:Xmmol.min-1

Enzymeunit酶的單位,SIunitTheamountthatcatalyzestheformationofonemmolofproductperminute每分鐘催化1mmol產物的形成所需的酶量U:mmol.min-1Katal,SIunitAmountofenzymecatalyzingtheconversionofonemoleofsubstratetoproductpersecond每秒鐘催化轉化1摩爾底物所需的酶量Katal:mol.s-1Expressionofenzymeactivity55Expressionofenzymeactivity酶活性的表示

11Expressionofenzymeactivity酶56Someexpressionsofthecomponentsofanenzyme酶的成分的幾種表示方式Enzyme酶Proteinpart+Cofactors(Non-proteinpart)(蛋白部分+輔助因子(非蛋白部分))Holoenzyme全酶Apoenzyme+Prostheticgroup(脫輔基酶+輔基)Someexpressionsofthecompon57Cofactors輔助因子InorganicInorganicion(s)

無機離子Fe2+,Zn2+,Cu2+,etc.Inorganiccluster(s)無機簇Fe-Scluster,Mn-Ocomplex,etcOrganicCoenzyme輔酶Whichlooselybindtotheirenzymes

Prostheticgroup輔基WhichtightlybindtotheirenzymesCofactors輔助因子Inorganic58Theimportantcoenzymesandprostheticgroups重要的輔酶和輔基Coenzymes輔酶CoenzymeA(CoA)NAD(H),NADP(H)Nicotinamideadeninedinucleotide煙酰胺腺嘌呤二核苷酸NicotinamideadeninedinucleotidephosphateProstheticgroups輔基FAD(H2),FMN(H2)*Flavinadeninedinucleotide黃素腺嘌呤二核苷酸Flavinmononucleotide黃素單核苷酸Biotin生物素Heme血紅素*FADandFMNinmostcasesareprostheticgroupsbindingtoflavinproteinsTheimportantcoenzymesandpr59Watersolublevitamins(VitaminB)Thiamine(B1)硫胺素InCoenzymes：thiaminepyrophosphate存在于硫胺焦磷酸輔酶中Riboflavin(核黃素B2)Asprostheticgroupflavinprotein:FADandFMNNicotinicacid(niacin煙堿酸)InCoenzymes:NADandNADPPantothenicacid(泛酸）InCoenzymeABiotin(生物素）Inprostheticgroup:Biocytin生物胞素Lipidsolublevitamins(VitaminA,D,E,K)VitaminARetinal視網膜素：vision,growthandreproductionMostcoenzymesarederivedfromvitamins

大多數輔酶源于維生素Watersolublevitamins(Vitami60Somecommoncoenzymes,theirvitaminprecursorsanddeficiencydiseasesCoenzyme/prostheticgroupPrecursorDeficiencydiseaseCoenzymeAPanthothenicacidDermatitis皮膚炎FAD,FMNRiboflavin(VitaminB2)GrowthretardationNAD+

(CoenzymeI),NADP+

(CoenzymeII)Niacin(菸堿酸)Pellagra玉蜀黍疹ThiaminepyrophosphateThiamine(VitaminB1)Beriberi腳氣病TetrahydrofolateFolicacidAnemia貧血癥Deoxyadenosylcobalamin（脫氧腺苷鈷胺素）Cobalamin(VitaminB12)Perniciousanemia惡性貧血癥Co-substrateinthehydroxylationofprolineincollagenVitaminC(ascorbicacid)Scurvy壞血病Pyridoxalphosphate磷酸吡哆醛Pyridoxine(vitaminB6)Dermatitis皮膚炎Somecommoncoenzymes,theirv61CoenzymeATransferofacetylgroupCoenzymeATransferofacetylg62NAD+,NADP+CoenzymeofDehydrogenasesNAD+,NADP+Coenzymeof63NAD(P)++H++2e-NAD(P)HReductionOxdationNAD+,NADP+H++2e-H-NAD(P)++H-NAD(P)HReductionOxdationGeneralreactionNAD(P)++H++2e-NAD(P)HRe64FAD,FMNFig.18.21,p591Isoalloxazine異咯嗪Flavinmononucleotide,FMNFlavinadeninedinucleotide,FADRiboflavinAMPRibitol核醣醇FAD,FMNFig.18.21,p591Isoall65FAD,FMNFAD+2H++2e-FADH2ReductionOxdationFMN+2H++2e-FMNH2ReductionOxdationFAD,FMNFAD+2H++2e-FADH66ChemicalstructureofbiotinChemicalstructureofbiotin67Chapter14EnzymekineticsIntroductiontoenzymes

酶（學）導論Factorsaffectingenzymeactivity

影響酶活性的因素EnzymeassaysEnzymekineticsandinhibition

酶動力學和抑制作用Chapter14EnzymekineticsIn68Cofactors輔助因子(SEEthelastsection)

pHTemperatureSubstrateconcentration底物濃度ProductconcentrationFactorsaffectingenzymeactivity

Chapter14EnzymesCofactors輔助因子(SEEthelasts69EffectofpH

Pepsin胃蛋白酶Papain木瓜蛋白酶Trypsin胰蛋白酶Cholinesterase膽堿酯酶過氧化氫酶EffectofpHPepsinPapain木瓜蛋白酶T70Effectoftemperature<40°C,increasewithriseoftemperature>50°C,decreaseduetodenaturationofenzymeBATypicallyEffectoftemperature<40°C,in71Enzymeassays酶的檢測TwomostconvenientwaysDetg.therateofappearanceofproduct測定產物形成的速率Detg.therateofdisappearanceofsubstrate測定反應物減少的速率Alinkedenzymeassayway酶聯檢測法Firstenzymereaction+secondenzymereactionMeasurements測定Spectrophotometry分光光度法Enzymeelectrode酶電極法Enzymeassays酶的檢測Twomostcon72LDH:lactatedehydrogenase乳酸脫氫酶COO-CHOHCH3+NAD+COO-COCH3Pyruvate丙酮酸Lactate乳酸+H+NADH+A340nmLDHLDH:lactatedehydrogenase乳酸73葡萄糖氧化酶過氧化氫氧化酶葡萄糖酸葡萄糖氧化酶過氧化氫氧化酶葡萄糖酸74Chapter14EnzymekineticsIntroductiontoenzymes

酶（學）導論Factorsaffectingenzymeactivity

影響酶活性的因素EnzymeassaysEnzymekineticsandinhibition

酶動力學和抑制作用Chapter14EnzymekineticsIn75Enzymekineticsandinhibition

(酶動力學和抑制作用)Catalyticmechanism催化機制Enzymekinetics酶動力學Michaelis-MentenmodelEnzymeinhibition酶的抑制Reversibleinhibition可逆抑制Nonreversibleinhibition非可逆抑制Enzymekineticsandinhibition76EnzymescatalyzechemicalreactionthroughloweringitsactivationenergyG<0:thermodynamicallyfavorableG>0:notthermodynamicallyfavorableProgressofreactionFreeenergy,GTransitionstate(uncatalyzed)Transitionstate(catalyzed)G?uncatalyzedG?catalyzedSPGEnzymescatalyzechemicalreac77ActivationEnergy~RateConstantofReactionG?~k

Arrheniusequationk=Ae-G?/RTG?kActivationEnergy~RateConst78Questions(selfstudy)