Substrate Activation of Trypsin and Acetyltrypsin Caused by α-N-Benzoyl-L-arginine Ρ-Nitroanilide*

Abstract

The initial rates of hydrolysis of α-N-benzoyl-L-arginine p-nitroanilide catalyzed by bovine trypsin [EC 3. 4. 4. 4] were examined with a wide range of substrate concentrations. The rates at high substrate concentrations were found to be exceedingly higher than those expected from the simple Michaelis-Menten equation which seemed applicable at the lower concentration. The phenomenon is observed not only with the commercial enzyme preparation but also with isolated α- or β-trypsin. The kinetics can be fitted to the substrate activation scheme, which has been proposed by Trowbridge etal. to account for their findings on a similar anomalous behavior of trypsin with α-N-tosyl-L-arginine methyl ester as substrate. The activity of trypsin toward the same nitroanilide substrate is enhanced significantly by acetylation of the enzyme, especially on its tyrosyl residues with acetyl-imidazole. The enhancement is prominent only at the low substrate concentration. The acetylation of trypsin has been reported to intensify the activity toward α-N-tosyl-L-arginine methyl ester only at high substrate concentrations and to amplify the substrate activation in the enzyme. The striking contrast observed between the two substrates may reflect the difference in the rate limiting step of their hydrolyses catalyzed by trypsin.