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1、Chapter 2 Enzymes,Reduced DsbA from E.coli,Lysozyme,3/28/2023,Enzyme,2,2.1 Introduction,Definition,History,Bchner,1897:A breakthrough in Enzymology.Catalysts at work in a living organism could also function completely independently of any life process.Efforts to isolate and purify individual enzymes
2、 were Bchners discoveries.Sumner,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.,3/28/2023,Enzyme,3,Characteristics,Three-dimensional structure of the folded protein,determined by the sequence of th
3、e amino acids.,Fragile:mild temperature,pressure,pH,ion strength(ambient conditions).,Lower the activation energy of the reaction,but,3/28/2023,Enzyme,6,Enzymes are named by adding the suffix ase to the:End of the substrateSuch as ureaseThe reaction catalyzedSuch as alcohol dehydrogenase,Nomenclatur
4、e,Enzymes using familiar names:Pepsin in the digestive tractTrypsin in the digestive tractRennin used in cheese making“Old yellow”,which caused browning of sliced apples,3/28/2023,Enzyme,7,EC(Enzyme Commission)SIX classes numbered in FOUR digits,Classification,The first digitalThe second digital The
5、 third digital The fourth digital,main classes,actual substance,3/28/2023,Enzyme,8,Oxidoreductases,First digit 1 the class oxidoreductases.Second digit the donor of hydrogen atom or electron involved.AlcoholAldehyde or ketoneAlkene CH=CH-Primary amineSecondary amineNADH,NADPHThird digit hydrogen ato
6、m or electron acceptor.NAD+,NADP+Fe3+O2Otherwise unclassifiedFourth digit number for further identification.,3/28/2023,Enzyme,9,Transferases,First digit 2 the class transferases.Second digit general type of groups transferred.1-carbon groupAldehyde or ketoneAcyl group(-CO-R-)Glycosyl groupPhosphate
7、groupSulphur containing groupThird 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+A,3/28/2023,Enzyme,10,Hydrolases,First digit 3 the class hydrolases.Second digit the type of bond
8、 hydrolyzedEster Glycosidic PeptideOther C-N bondsAcid anhydrides,Hydrolyases catalyze hydrolytic reactions,with a general form given below:A-X+H2O X-OH+HA,3/28/2023,Enzyme,11,Lyases,First digit 4 the class lyases.Second digit the type of binds broken.C-CC-OC-NC-SThird digit The group removed.Carbox
9、ylAldehydeKeto acid Fourth digit number for further identification.,Lyases catalyze the non-hydrolytic removal of groups from substances.Often the product contains a double bond.,3/28/2023,Enzyme,12,Isomerases,First digit 5 the class isomerases.Second digit the type of reaction involved.Racemization
10、 or epimerizationCis-trans isomerizationIntramolecular oxidoreductasesIntramolecular transfer reactionsThird digit the type of molecule undergoing isomerization.Amino acidsHydroxyacidscarbohydratesFourth digit number for further identification.,3/28/2023,Enzyme,13,Ligases,First digit 6 the class lig
11、ases.Second 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+PiX+Y+ATP X-Y+AMP+PPi,3/28/2023,Enzyme,14,Some Exampl
12、es of Enzyme,Alcohol DehydrogenaseEC 1.1.1.1Glucose OxidaseEC 1.1.3.4CatalaseEC 1.11.1.6Tryptophan 2,3-dioxygenaseEC 1.13.11.11Pyruvate Kinase EC 2.7.1.40Creatine KinaseEC 2.7.3.2Alpha-amylaseEC 3.2.1.1ChitinaseEC 3.2.1.14Oxaloacetate DecarboxylaseEC 4.1.1.3Lactate RacemaseEC 5.1.2.1Ribose Isomerase
13、EC 5.3.1.20AcetateCoA LigaseEC 6.2.1.1Glutathione Synthase EC 6.3.2.3,3/28/2023,Enzyme,15,2.2 How Enzymes Work?,Lock and Key Model Developed by Emil Fischer in 1895.The enzymes and substrates combine because they have complementary molecular geometries.,3/28/2023,Enzyme,16,An Example of Lock-Key Mod
14、el,3/28/2023,Enzyme,17,Induced-Fit Model Like a hand and glove.The enzyme is a molecule whose conformation can change as the substrate approaches and starts to bind.,3/28/2023,Enzyme,18,An Example of Induced-Fit Model,3/28/2023,Enzyme,19,2.3 Enzyme Kinetics,Two assumptions:,Reaction occurred in well
15、-mixed reactor.That is to say,spatially uniform.Only initial rate is used:,which has the unit of M/s.,3/28/2023,Enzyme,20,2.3.1 Mechanistic Models for Simple Enzyme Kinetics,Single-substrate kinetics was first developed:V.C.R.Henri in 1902L.Michaelis and M.L.Menten in 1913,A simple reaction scheme:,
16、Saturation kinetics can be obtained for the reaction scheme above.,3/28/2023,Enzyme,21,The same few initial steps in deriving a rate expression:,3/28/2023,Enzyme,22,Rapid Equilibrium Assumption(Developed by Henri and Michaelis and Menten),ASSUMPTION:A rapid equilibrium between the enzyme and the sub
17、strate can be achieved to form an ES complex.,3/28/2023,Enzyme,23,A low value of suggests that the enzyme has a high affinity for the substrate.,3/28/2023,Enzyme,24,Experimental data demonstrated the concentration profiles.,ASSUMPTION:Initial substrate concentration greatly exceeds the initial enzym
18、e concentration.,is small,then,3/28/2023,Enzyme,25,3/28/2023,Enzyme,26,Both Give Saturation Kinetics,Saturation 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 con
19、centrations.,3/28/2023,Enzyme,27,Questions,Why only initial rate can be used?,Why a low value of suggests that the enzyme has a high affinity for the substrate?,3/28/2023,Enzyme,28,2.3.2 Experimentally Determining Rate Parameters for Michaelis-Menten Type Kinetics,With known S0 and E0,we can calcula
20、te the initial rate:,3/28/2023,Enzyme,29,Double-Reciprocal Plot(Lineweaver-Burk Plot),By rearrangement,we can get,3/28/2023,Enzyme,30,Eadie-Hofstee Plot,By rearrangement,we can also get,3/28/2023,Enzyme,31,Hanes-Woolf Plot,By rearrangement,we can also get,3/28/2023,Enzyme,32,Batch Kinetics,The time course of variation of S in a batch enzymatic reaction can be determined from,By integration to yield,or,A plot of versus results in a line ofslope and intercept of.,
