Open AccessStudies on the Catalytic Mechanism of a Serine Protease from Streptomyces griseus
Author(s) -
Bauer CarlAxel,
Löfqvist Bo,
Pettersson Gösta
Publication year - 1974
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1974.tb03242.x
Subject(s) - streptomyces griseus , chemistry , methanol , kinetics , catalysis , nucleophile , protease , reaction rate constant , dissociation (chemistry) , hydrolysis , rate determining step , reaction rate , substrate (aquarium) , serine protease , stereochemistry , enzyme , medicinal chemistry , streptomyces , organic chemistry , biology , ecology , genetics , physics , quantum mechanics , bacteria
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.