Determination of the binding constant of a specific ester and a specific amide substrate to α-chymotrypsin

Abstract

A transient-phase method is described for obtaining values for the individual rate constants of a single-substrate two-intermediate enzyme mechanism. In principle, values of k1, k−1and k3 can be obtained from such studies, as can values of inhibition constants kq and k−q from studies of a competitively-inhibited reaction. An experimental stopped-flow study is made for the α-chymotrypsin-catalyzed hydrolysis of p-nitrophenyl acetate, in the absence and presence of the inhibitor indole, and the results are consistent with the theoretical predictions. Values of k1, k−1, k2and k3 were obtained.The transient-phase equations for many enzyme systems, at limiting enzyme concentration, are in all cases of the form. where n, the number of exponential terms, is equal to the number of enzyme-containing intermediates in the mechanism (not counting the free enzyme). The sum of the exponents λ, i.e. ∑i=1n, is equal to the sum of all of the first-order rate constants, plus the sum of all the second-order rate constants each multiplied by the corresponding substrate or modifier concentration: where ki is a second-order rate constant and kj a first-order constant. This suggests general procedures for obtaining rate constants. Published results on α-chymotrypsin and alkaline phosphatase are reinterpreted in the light of the theory.