Reactivity and Interrelationship of Intermediates in the Hydrolysis of p-Nitrophenyl Acetate Catalysed by Chymotrypsin

Abstract

KINETIC studies by Gutfreund and Sturtevant¹ suggest that p-nitrophenyl acetate reacts with chymotrypsin rapidly at a pH greater than 6.5 to form monoacetyl chymotrypsin (AC–I) and that AC–I is then deacylated slowly. The kinetic observations are consistent with the esterification of a single serine hydroxyl of the enzyme². In contrast, spectroscopic studies³ of the deacylation of monoacetyl chymotrypsin formed at pH 5.0 and isolated according to the procedure of Balls and Wood⁴, (AC–A), show that when AC–A is brought to pH 9.0, a rapid increase in absorption at 245 mµ occurs which is followed by slow decay of the absorption peak. Since both the absorption peak and its rate of decay appeared characteristic for N-acetyl imidazole, Dixon and Neurath³ suggested that the deacylation of monoacetyl chymotrypsin proceeds by a rapid intramolecular shift of the acetyl group from a serine oxygen to an imidazole nitrogen of the enzyme (AC–II), and that the rate-limiting step of the enzymatic reaction is the base-catalysed hydrolysis of N-acetyl imidazole. Recently, however, we have observed⁵ that AC–I and AC–Aare deacylated at different rates as measured by the liberation of p-nitrophenol from p-nitrophenyl acetate catalysed by AC–I and AC–A, and that AC–I is converted to AC–A under the conditions used in the isolation procedure⁴.