细胞生物化学实验课件Exp-3-Determination-of-Km-Lei-Zhang-upd.ppt

1、细胞生物化学实验课件Exp 3-Determination of Km-Lei Zhang-updatedOverview Objective Principle Procedure Results and analysis Notes Thought questionsObjective 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.Prin

2、ciples The stages of an enzyme-catalysed reaction are summarised as E+S ES EP E+PMichaelis-Menten Equation V=initail velocity of the reaction Vmax=maximum velocity of the reaction S=substrate concentration Km=Michaelis constantS+KSV=VmmaxMichaelis Constant(Km)The Km value for an enzyme depends on pa

3、rticular substrate 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/2 Then,at V=Vmax/2,Km=S Thus,the unit of Km is mol/L,just as SS+

4、KSV=VmmaxHyperbolaAt low 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

5、 plateau.The reaction becomes 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 amoun

6、t and the turnover number of the enzyme.Lineweaver-Burk Double-reciprocal PlotSVS+SVK=V1maxmaxmS+KSV=VmmaxmaxmaxmV1+S1VK=V1SVS+K=V1maxm When used for determining the type of enzyme inhibition,the LineweaverBurk plot can distinguish competitive and non-competitive inhibitors of enzyme.Alkaline Phosph

7、atase(AKP)Alkaline phosphatases(AKP)are the phosphate hydrolases that have a maximum 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 d

8、isodium phenylphosphate is used to measure the activity of AKP.Disodium phenylphosphate 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-aminoant

9、ipyrine can be oxidized to quinone derivatives.The more phenol active as substrate,the more quinone derivatives are produced,which are red compounds with maximum absorption peak at 510nm.ProcedureThe impact of substrates concentration on velocity of enzyme reaction (1)Take 6 test tubes and label the

10、m as 1-6.(without KH2PO4)(2)mix test tubes 1-6 well and incubate at 37 for 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)

11、#6 and 6 tubes are as blank.Measure the A510 of each samples using the spectrophotometer 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

12、and incubate at RT for 15 minutes.Measure the A510 of samples and the blank tube is used for the zero setting.(3)A510 is plotted against phenol concentration.Results and analysis1.Figure of reaction kinetics(1/A510 vs.1/S)2.Value of Km 3.Enzyme activity One unit of enzyme activity is defined as the

13、amount that catalyzes the formation of one milligram of product(phenol)in 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.