《细胞生物化学实验ppt课件Exp-3-Determination-of-Km--Lei-Zhang-upd.ppt》由会员分享，可在线阅读，更多相关《细胞生物化学实验ppt课件Exp-3-Determination-of-Km--Lei-Zhang-upd.ppt（18页珍藏版）》请在三一办公上搜索。
1、细胞生物化学实验课件Exp 3-Determination of Km-Lei Zhang-updated,Overview,ObjectivePrincipleProcedureResults and analysisNotesThought questions,Objective,To understand the significance of Km and learn how to determine the Km value of AKP.To be able to calculate the Km of enzyme using the standard curve.,Princi
2、ples,The stages of an enzyme-catalysed reaction are summarised asE+S ES EP E+P,Michaelis-Menten Equation,V=initail velocity of the reactionVmax=maximum velocity of the reactionS=substrate concentrationKm=Michaelis constant,Michaelis Constant(Km),The Km value for an enzyme depends on particular subst
3、rate and on the environmental conditions,such as temperature,pH,and ionic strength,regardless of enzyme concentration.The Km value is a characteristic constant of enzymes.,Plot of Michaelis-Menten Equation,When S=Km,V=Vmax/2Then,at V=Vmax/2,Km=SThus,the unit of Km is mol/L,just as S,Hyperbola,At low
4、 concentrations of the substrate S,velocity(V)is proportional to S,is a usual features of a first order reaction.With the increase of substrate concentration,V does not increase proportionally to S.At substrate concentrations far higher than Km,(SKm),the curve approaches to a plateau.The reaction be
5、comes nearly independent of the substrate concentration and shows zero-order kinetics.When the enzyme is saturated by substrate,almost all enzyme molecules are present as enzyme-substrate complex and the reaction is no longer limited by substrate availability but by the amount and the turnover numbe
6、r of the enzyme.,Lineweaver-Burk Double-reciprocal Plot,When used for determining the type of enzyme inhibition,the LineweaverBurk plot can distinguish competitive and non-competitive inhibitors of enzyme.,Alkaline Phosphatase(AKP),Alkaline phosphatases(AKP)are the phosphate hydrolases that have a m
7、aximum activity at a relatively high pH(7.0).AKP is widespread and occurs in both eukaryotic and prokaryotic cells.Some different substrates can be catalyzed by AKP with different Km.,In this experiment,a substrate called disodium phenylphosphate is used to measure the activity of AKP.Disodium pheny
8、lphosphate can be hydrolyzed by AKP and produce phenol and phosphates.The higher activity of AKP the more phenol is produced.So the concentration of phenol varies in proportion with the activity of AKP.Phenol and 4-aminoantipyrine can be oxidized to quinone derivatives.The more phenol active as subs
9、trate,the more quinone derivatives are produced,which are red compounds with maximum absorption peak at 510nm.,Procedure,The impact of substrates concentration on velocity of enzyme reaction(1)Take 6 test tubes and label them as 1-6.(without KH2PO4),(2)mix test tubes 1-6 well and incubate at 37 for
10、15 minutes.(3)add 1.1ml of alkaline solution into each tube to terminate reaction.(4)add 1.0ml of 0.3%4-aminoantipyrine and 2.0ml of 0.5%potassium ferricyanide into each tubes,mix well then place them at RT for 10 minutes.(5)#6 and 6 tubes are as blank.Measure the A510 of each samples using the spec
11、trophotometer and the blank tube is used for the zero setting.(6)Plot 1/A510 against 1/S and calculate Km.,Plot a standard curve of phenol contentTake 6 test tubes and label them as s1-s6，#s1 tube is used as blank.(2)mix well and incubate at RT for 15 minutes.Measure the A510 of samples and the blan
12、k tube is used for the zero setting.(3)A510 is plotted against phenol concentration.,Results and analysis,1.Figure of reaction kinetics(1/A510 vs.1/S)2.Value of Km 3.Enzyme activityOne unit of enzyme activity is defined as the amount that catalyzes the formation of one milligram of product(phenol)in
13、 15 minutes at 37.Calculate the enzyme activity according to the standard curve of phenol content.,Notes,Pipetting the reagents should be accurate and correct.Standard curve must be a straight line across base point(0,0).,Thought questions,What is the significance of measuring Km?Compare the differences among three reversible inhibitions.,
