Kinetics of hydrolysis of amide and anilide substrates of p-guanidino-L-phenylalanine by bovine and porcine trypsins.

The rates of hydrolysis of N^α-benzoyl-p-guanidine-L-phenylalaninamide (Bz-GPA-NH₂) and N^α-substituted p-nitroanilides (pNA) of GPA (benzyloxycarbonyl(Z)-GPA-pNA, benzoyl(Bz)-GPA-pNA and acetyl(Ac)-GPA-pNA) by bovine and porcine trypsins were compared with those of arginine (Arg) substrates. The amide type substrates of GPA were hydrolyzed as fast as those of Arg by the two enzymes with much the same k_cat/K_m values, though significant differences were found between the k_cat and K_m values of GPA derivatives and those of Arg derivatives. The kinetic behavior of porcine trypsin toward GPA substrates was almost the same as that of the bovine enzyme. The ratio of the k_cat value for Bz-GPA-OEt to that for Bz-GPA-NH₂ was much larger than that for the ester to amide substrates of arginine, suggesting that the conformational change of the active site of trypsin induced by a benzene ring in the side chain of Bz-GPA-OEt specifically increases the velocity of the deacylation process of the ester substrate. Remarkably low values of both kcat and Km were found for the tryptic hydrolysis of Z-GPA-pNA and Ac-GPA-pNA, as well as on that of Bz-GPA-pNA (Tsunematsu, H., et al. (1983) J. Biochem. 94, 123-128). Z-GPA-pNA is the best substrate for the two trypsins among the three N^α-substituted anilide substrates of GPA. Substrate activation was observed with bovine trypsin in the hydrolysis of the three anilide substrates of GPA in a substrate concentration range higher than about 5.0×10^-4M, but it was found with the porcine enzyme only in the hydrolysis of Z-GPA-pNA. In contrast, no activation by the amide substrate of GPA could be detected with either enzyme.