Studies on the Catalytic Mechanism of a Serine Protease from Streptomyces griseus

The kinetics of the Streptomyces griseus protease‐3‐catalyzed hydrolysis of p ‐nitrophenyl‐acetate have been studied by stopped‐flow techniques with widely varied initial concentrations of enzyme and substrate. The results obtained are consistent with a three‐step mechanism in which there is a rapid equilibration between enzyme and substrate to form a Michaelis complex with a dissociation constant in the order of 10 mM, followed by a moderately rapid formation of an acyl‐enzyme and a rate‐limiting deacylation of this intermediate. Estimated rate constants forthe latter two reaction steps are 15 s −1 and 0.09 s −1 , respectively. The presence of a nucleophile such as methanol results in a partitioning of the deacylation process, reflected by a linear dependence of the maximum steady‐state reaction velocity on the concentration of methanol. This observation provides confirmatory evidence for a rate‐limiting deacylation step in the catalytic mechanism.