Kinetic Study on the Binding of 1,1,1-Trichloro-2,2-bis(4-chlorophenyl)ethane to Rhizopus delemar C-Lipase : Rate Constants and the Effect of Slow Addition of the Ligand on Complex Formation

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Rhizopus delemar C-lipase (E) binds 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (D or DDT) to form stable 1 : 1,2 : 1 and 9 : 1 DDT-lipase complexes as follows.[table]Competitive binding experiments with a mixture of E and ED and that of E and E'D_2 yielded κ_1 : κ_2 : κ_4=80 : 1 : 400. The κ_2 value was estimated to be 1.6×10^8 M^<-1>s^<-1> by following the decrease in [ED] at a low concentration of reactants, ED and DDT, where the reaction was terminated at a given time by scavenging DDT with the addition of excess E'D_2. The κ_3 value, 0.021s^<-1>, was determined by follwoing the formation of E'D_2 from ED_2 in the presence of ED. E'D_2 was estimated by converting E'D_2 rapidly into E^*D_9 by adding an adequate amount of DDT (ED_2 cannont bind DDT), and the excess free DDT was depleted by the pre-existing ED to stop E^*D_9 formation from newly born E'D_2.The continuous slow addition of a limited amount of DDT to ED made the rate of the second order reaction, ED to ED_2,comparable to the rate of the slow first order reaction, ED_2 to E'D_2,thereby favoring the formation of E^*D_9. The estimation of κ_2 and κ_3 from the variation in the final population of each DDT-lipase species as a function of the rate of ligand addition was described.
社団法人日本薬学会の論文
1992-12-25