生物反应工程：chap2enzyme-课件.ppt

1、12/15/2022Enzyme1Reduced DsbA from E.coliLysozyme12/15/2022Enzyme2DefinitionHistoryuBchner,1897:A breakthrough in Enzymology.lCatalysts at work in a living organism could also function completely independently of any life process.lEfforts to isolate and purify individual enzymes were Bchners discove

2、ries.uSumner,1926:First isolation of a pure enzyme.Enzymes are usually proteins of high molecular weight(15,000 MW several million Daltons)that act as catalysts.12/15/2022Enzyme3CharacteristicsuThree-dimensional structure of the folded protein,determined by the sequence of the amino acids.uFragile:m

3、ild temperature,pressure,pH,ion strength(ambient conditions).uLower the activation energy of the reaction,butDoesnt affectFree-energy changeEquilibrium constant12/15/2022Enzyme6uEnzymes are named by adding the suffix ase to the:lEnd of the substrateSuch as ureaselThe reaction catalyzedSuch as alcoho

4、l dehydrogenaseNomenclatureuEnzymes using familiar names:lPepsin in the digestive tractlTrypsin in the digestive tractlRennin used in cheese makingl“Old yellow”,which caused browning of sliced apples12/15/2022Enzyme7uEC(Enzyme Commission)uSIX classes numbered in FOUR digitsClassificationThe first di

5、gitalThe second digital The third digital The fourth digitalType of reaction catalyzed main classes actual substance12/15/2022Enzyme8uOxidoreductaseslFirst digit 1 the class oxidoreductases.lSecond digit the donor of hydrogen atom or electron involved.AlcoholAldehyde or ketoneAlkene CH=CH-Primary am

6、ineSecondary amineNADH,NADPHlThird digit hydrogen atom or electron acceptor.NAD+,NADP+Fe3+O2Otherwise unclassifiedlFourth digit number for further identification.12/15/2022Enzyme9uTransferaseslFirst digit 2 the class transferases.lSecond digit general type of groups transferred.1-carbon groupAldehyd

7、e or ketoneAcyl group(-CO-R-)Glycosyl groupPhosphate groupSulphur containing grouplThird digit provide details on the exact name of the group transferred.Transferases catalyze the functional group transfer reactions,with a general form given below:AX+B BX+A12/15/2022Enzyme10uHydrolaseslFirst digit 3

8、 the class hydrolases.lSecond digit the type of bond hydrolyzedEster Glycosidic PeptideOther C-N bondsAcid anhydridesHydrolyases catalyze hydrolytic reactions,with a general form given below:A-X+H2O X-OH+HA12/15/2022Enzyme11uLyaseslFirst digit 4 the class lyases.lSecond digit the type of binds broke

9、n.C-CC-OC-NC-SlThird digit The group removed.CarboxylAldehydeKeto acid lFourth digit number for further identification.Lyases catalyze the non-hydrolytic removal of groups from substances.Often the product contains a double bond.12/15/2022Enzyme12uIsomeraseslFirst digit 5 the class isomerases.lSecon

10、d digit the type of reaction involved.Racemization or epimerizationCis-trans isomerizationIntramolecular oxidoreductasesIntramolecular transfer reactionslThird digit the type of molecule undergoing isomerization.Amino acidsHydroxyacidscarbohydrateslFourth digit number for further identification.12/1

11、5/2022Enzyme13uLigaseslFirst digit 6 the class ligases.lSecond digit the type of bonds formed.C-OC-SC-NC-C Ligases catalyze the synthesis of various types of bonds,where the reactions are coupled with breakdown of energy-containing materials,such as ATP or nucleoside triphosphates.X+Y+ATP X-Y+ADP+Pi

12、X+Y+ATP X-Y+AMP+PPi12/15/2022Enzyme14lAlcohol DehydrogenaseEC 1.1.1.1lGlucose OxidaseEC 1.1.3.4lCatalaseEC 1.11.1.6lTryptophan 2,3-dioxygenaseEC 1.13.11.11lPyruvate Kinase EC 2.7.1.40lCreatine KinaseEC 2.7.3.2lAlpha-amylaseEC 3.2.1.1lChitinaseEC 3.2.1.14lOxaloacetate DecarboxylaseEC 4.1.1.3lLactate

13、RacemaseEC 5.1.2.1lRibose IsomeraseEC 5.3.1.20lAcetateCoA LigaseEC 6.2.1.1lGlutathione Synthase EC 6.3.2.312/15/2022Enzyme15Lock and Key Model Developed by Emil Fischer in 1895.The enzymes and substrates combine because they have complementary molecular geometries.12/15/2022Enzyme1612/15/2022Enzyme1

14、712/15/2022Enzyme1812/15/2022Enzyme19Two assumptions:Reaction occurred in well-mixed reactor.That is to say,spatially uniform.Only initial rate is used:000 tttdtdSdtdPvTwo major approaches:Rapid equilibrium approach;Quasi-steady-state approach.which has the unit of M/s.12/15/2022Enzyme20Single-subst

15、rate kinetics was first developed:lV.C.R.Henri in 1902lL.Michaelis and M.L.Menten in 1913A simple reaction scheme:Saturation kinetics can be obtained for the reaction scheme above.12/15/2022Enzyme21The same few initial steps in deriving a rate expression:The rate of variation of the ES complex:211ES

16、kESkSEkdtESd Eq.(2.2)The conservation equation on the enzyme:Eq.(2.3)0ESEE The rate of product formation:2ESkdtPdv Eq.(2.1)12/15/2022Enzyme22Rapid Equilibrium Assumption(Developed by Henri and Michaelis and Menten)ASSUMPTION:A rapid equilibrium between the enzyme and the substrate can be achieved to

17、 form an ES complex.The dissociation constant:11ESSEkkKm Eq.(2.4)Substituting Eq.(2.3)into Eq.(2.4)gives,0SKSEESm Eq.(2.5)12/15/2022Enzyme23Substituting Eq.(2.5)into Eq.(2.1)yields,02SKSVSKSEkvmmm Eq.(2.6)where ,.11kkKm 02EkVm A low value of suggests that the enzyme has a high affinity for the subst

18、rate.mK mV the maximum forward velocity of the reaction;if the amount of the enzyme changes,changes the Michaelis-Menten constantmK mV12/15/2022Enzyme24Experimental data demonstrated the concentration profiles.ASSUMPTION:Initial substrate concentration greatly exceeds the initial enzyme concentratio

19、n.0E0 dtESdis small,thenThe Quasi-Steady-State Assumption(Developed by G.E.Briggs and J.B.S.Haldane)12/15/2022Enzyme25From Eq.(3.2)and the assumption,we have,211kkSEkES Eq.(2.7)Substituting Eq.(2.3)in to Eq.(2.7)gives1210SkkkSEES Eq.(2.8)Substituting Eq.(2.8)in to Eq.(2.1)yields20121 mmk ESVSvkkKSSk

20、Eq.(2.9)where ,.02EkVm 121kkkKm 12/15/2022Enzyme26Both Give Saturation KineticsSaturation kinetics,similar to Langmuir-Hinshelwood isothermal adsorption kinetics,which shows a first-order kinetics at the low substrate concentrations,but zero-order kinetics at high substrate concentrations.12/15/2022

21、Enzyme27Questions Why only initial rate can be used?Why a low value of suggests that the enzyme has a high affinity for the substrate?mK 12/15/2022Enzyme280t0tinitdtSddtPdv With known S0 and E0,we can calculate the initial rate:12/15/2022Enzyme29Double-Reciprocal Plot(Lineweaver-Burk Plot)By rearran

22、gement,we can getS1VKV1v1mmm 12/15/2022Enzyme30Eadie-Hofstee PlotBy rearrangement,we can also getSvKVvmm 12/15/2022Enzyme31Hanes-Woolf PlotBy rearrangement,we can also get1SVVKvSmmm12/15/2022Enzyme32Batch KineticsThe time course of variation of S in a batch enzymatic reaction can be determined from,SKSVdtSdvmm By integration to yield,ln00SSKSStVmm or,ln00SStKtSSVmm A plot of versus results in a line ofslope and intercept of .ln10SSttSS0 mK1 mmKV