The Mechanism of the α‐Chymotrypsin and Trypsin‐Catalyzed Hydrolysis of Amides

The applicability of the acylenzyme mechanism to the tryptic hydrolysis of the amide bond has been studied. It was found that trypsin acetylated at the hydroxyl group of the “active” serine or acetyl (Ser‐183)trypsin displays no activity with respect to both esters ( N ‐benzoyl‐ l ‐arginine ethyl ester and p ‐nitrophenyl‐ p′ ‐guanidinobenzoate) and anilides ( N ‐carbobenzoxy‐ l ‐arginine m ‐nitroanilide and N ‐benzoyl‐ dl ‐arginine p ‐nitroanilide). It was also found, that deacetylation of acetyl(Ser‐183)trypsin is effectively promoted by N ‐carbobenzoxy‐ dl ‐arginine m ‐nitroanilide (Z‐Arg‐Nan) taken at a high concentration. As was found by means of a detailed kinetic analysis of experimental results, the binding of this anilide with acetyl(Ser‐183)trypsin increases the rate constant of deacylation of the latter by as many as 23 times. This effect which results in a rapid increase in the concentration of native trypsin in the reaction system, causes a marked increase in both k cat and K m(app) of tryptic hydrolysis of Z‐Arg‐Nan). Apparently, the phenomena is the main reason for the erroneous conclusions of Bresler et al. (1969, 1971), who stated: trypsin with an acylated active serine remains a highly efficient catalyst of amide hydrolysis. Finally, the data of Bresler et al. on tryptic hydrolysis and hydroxylaminolysis which contradict the acylenzyme mechanism of hydrolysis of the amide bond can be readily accounted for by chemistry of hydroxylamine, as was done by Bender et al. (1964) and Epand and Wilson (1963). Thus, the data obtained so far strongly support the acyl‐enzyme mechanism of the hydrolysis of the amide bond catalyzed by trypsin and α‐chymotrypsin.