Trypsin-like serine proteinase action: determination of the catalytic parameters KS, k+2 and k/3 under conditions where the substrate exceeds the enzyme concentration

A method for the determination of the catalytic parameters Ks, k+2 and k+3 describing trypsin-like serine proteinase action has been developed from the quantitative analysis of the kinetics of hydrolysis of two specific chromogenic substrates, N-alpha-carbobenzoxy-L-arginine p-nitrophenyl ester and N-alpha-carbobenzoxy-L-lysine p-nitrophenyl ester, catalyzed by porcine pancreatic betta-kallikrein B, bovine betta-trypsin and human urokinase (Mr 54,000 species), under conditions where the concentration of the substrate exceeds that of the enzyme. Value of Ks, k+2 and k+3 have been estimated from the effect of substrate concentration on the apparent first-order rate constant of the time-course of the burst phase of p-nitrophenol release preceding the steady-state reaction.