The mechanism of the reaction of chymotrypsin with p-nitrophenyl acetate

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Abstract
The kinetics of the hydrolysis of p-nitrophenyl acetate catalyzed by chymotrypsin can be fully described by a mechanism involving 3 distinct steps. The 1st step, which involves the rapid adsorption of the substrate on the enzyme, is too fast to be measured by the methods used. The 2d step, which involves the acylation of the enzyme and the concomitant liberation of p-nitrophenol is characterized by the rate constant k2 = 3.15 sec.-1. The third step involves the liberation of acetate and reactivation of the enzyme; the rate of this reaction is defined by k3 = 0.0254 sec.-1. The rate constant k2 for the 2d step is, within experimental error, the same at pH 6.45 and pH 7.75; this and other available evidence suggest that it involves the acetylation of the OH group of a serine residue in the enzyme. The 3d step has a pH dependence similar to that for the overall rate of reactions catalyzed by chymotrypsin, and it is suggested that it involves the imidazole group of a histidine residue of the enzyme.