Kinetics of hydrolysis of Na-benzoyl-p-guanidino-L-phenylalanine p-nitroanilide by trypsin

Abstract

A new chromogenic substrate, Na-benzoyl-p-guanidino-L-phenylalanine p-nitroanilide (Bz-GPA-pNA), was synthesized. This compound is a good substrate for bovine trypsin (Km = 1.56 X 10(-5) M, kcat = 0.081 s -1, at pH 8.2) and was hydrolyzed as fast as Na-benzoyl-L-arginine p-nitroanilide (Bz-Arg-pNA) with much the same kcat/Km values. But the values are two orders of magnitude smaller than those for ester substrates, Na-benzoyl-p-guanidino-L-phenylalanine ethyl ester (Bz-GPA-OEt) and Na-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt). Substrate activation behavior was observed on tryptic hydrolysis of this new substrate in a substrate concentration range higher than about 5.0 X 10(-4) M in analogy with the trypsin-Bz-Arg-pNA system.