Locating the rate-determining step(s) for three-step hydrolase-catalyzed reactions with dynafit

Hydrolytic reactions of oligopeptide 4-nitroanilides catalyzed by human-α-thrombin, human activated protein C and human factor Xa were studied at pH 8.0–8.4 and 25.0 ± 0.1 °C by the progress curve method and individual rate constants were calculated mostly within 10% internal error using DYNAFITV. A systematic strategy has been developed for fitting a three-step consecutive mechanism to eighteen hundred to six thousand time-course data points polled from two to four independent kinetic experiments. Enzyme and substrate concentrations were also calculated. Individual rate constants well reproduce published values obtained under comparable conditions and the Michaelis–Menten kinetic parameters calculated from these elementary rate constants are also within reasonable limits of published values. For comparison, the integrated Michaelis–Menten equation was also fitted to data from twelve sets. Both the kcat and kcat/Km values are within 15% agreement with those calculated using the elementary rate constants obtained with DYNAFITV. Rate constants for the second and third consecutive steps are within 3–4 fold indicating that both determine the overall rate. The Factor Xa-catalyzed hydrolysis of N-α-Z-d-Arg-Gly-Arg-pNA · 2HCl at pH 8.4 in a series of buffers containing increasing fractions of deuterium at 25.0 ± 0.1 °C shows a very strong dependence of k3 and a moderate dependence of k2 on D content in the buffer: the fractionation factors are: 0.49 ± 0.03 for K1, 0.70 ± 0.05 for k2, and (0.32 ± 0.03)2 for k3.