Two-step interaction of alpha-chymotrypsin with a fluorescent inhibitor. A dynamic study by the temperature-jump...

Bovine alpha-chymotrypsin interacts with a dansylated inhibitor, 1-dimethylaminonaphthalene-5-sulfonyl-D-tryptophan ethyl ester, to give a stable and strongly fluorescent derivative. The kinetics of the interaction of the enzyme with this inhibitor has been investigated by the temperature-jump method. It is shown that the complex formation proceeds in two steps, a binding step and a slow isomerisation, as deduced from a study of the concentration dependence of the relaxation times. The rate constants for the binding process are k12 = 7.9 x 10(5) M-1s-1 and k21 = 1.36 x 10(3) s-1, giving a binding constant of 585 M-1. This last value, compared to the overall binding constant determined statically of 1740 M-1, leads to the determination of the kinetic parameters for the slow isomerisation k23 = 19 s-1 and k32 = 9 s-1. Both values have the same order of magnitude as the rate constants found for the pH-induced relaxation of some pancreatic proteins. This slow isomerisation can be related to internal motions of the active center of the enzyme.