EFFECTS OF ACETYLATION UPON ACTIVITY OF TRYPSIN TOWARD ESTER AND AMIDE SUBSTRATES

The activities of crystalline bovine trypsin toward simple amide and ester substrates are enhanced significantly by acetylation of the enzyme with 0.1 m M-acetylimidazole at pH 7.5. Neither the presence of 0.14 m α-N-p-toluenesulfonyl-L-arginin amide nor that of 5 × 10-3 m benzamidine diminishes the degree of enhancement, suggesting that the important modifications do not involve aminoacyl residues of the active site. Acylation of exposed tyrosyl residues appears to be fundamental to the increase in activity, which can be reversed by deacylation with 0.5 m hydroxylamine (pH 7.5). The increased activity toward specific, low molecular weight amide substrates is independent of the nature of the α-amino blocking group (p-toluene-sulfonyl us. benzoyl) on the substrate. No evidence of substrate activation is observed in the concentration range employed (10-4-10-1 m). The magnitude of the enhancement of esterolytic activity is strongly dependent upon substrate concentration and upon the nature of the -amino blocking group, being dramatic with concentrations of α-N-p-toluenesulfonyl-L-arginine methyl ester greater than 10-4 m and very modest at all concentrations of α-N-benzoyl-L-arginine ethyl ester. Steady-state kinetic studies related the enhanced activities primarily to increased catalytic constants. Enhancement of this operational factor could result either from an increase in the rate constant for the rate-determining step or from an increase in the concentration of the productive form of the intermediate consumed in this step due to an altered conformational and/or protonic equilibrium. © 1969, American Chemical Society. All rights reserved.