1992 Volume 40 Issue 12 Pages 3297-3302
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'D2 yielded κ1 : κ2 : κ4=80 : 1 : 400. The κ2 value was estimated to be 1.6×108 M-1s-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'D2. The κ3 value, 0.021s-1, was determined by follwoing the formation of E'D2 from ED2 in the presence of ED. E'D2 was estimated by converting E'D2 rapidly into E*D9 by adding an adequate amount of DDT (ED2 cannont bind DDT), and the excess free DDT was depleted by the pre-existing ED to stop E*D9 formation from newly born E'D2.The continuous slow addition of a limited amount of DDT to ED made the rate of the second order reaction, ED to ED2, comparable to the rate of the slow first order reaction, ED2 to E'D2, thereby favoring the formation of E*D9. The estimation of κ2 and κ3 from the variation in the final population of each DDT-lipase species as a function of the rate of ligand addition was described.
