BOVINE FACTOR XA AND BOVINE TRYPSIN - COMPARISON WITH RESPECT TO INHIBITION BY SOME AMIDINES AND GUANIDINES

The enzymatic activity of activated bovine blood clotting factor X [EC 3.4.21.6] toward the synthetic substrate N .alpha.-benzoyl-L-arginine ethyl ester and the inhibitory effects of a series of low MW synthetic aromatic amidine and guanidine compounds on that activity were studied using the steady-state kinetic method. The kinetic parameters, Km and kcat, and the apparent dissociation constant Ki'' for each inhibitor, were determined for activated factor X hydrolysis of Bz-Arg-OEt at 37.degree. C, pH 7.8 in 0.1 N NaCl and 0.001 M CaCl2. The same constants were determined for bovine .beta.-trypsin [EC 3.4.21.4] under identical conditions. Comparison of kinetic constants determined for both enzymes shows that activated factor X binds the substrate Bz-Arg-OEt less efficiently than .beta.-trypsin by several orders of magnitude. Binding of the inhibitors benzamidine, p-aminobenzamidine, pentamidine, M&B 4596, phenylguanidine and p-guanidinobenzoic acid is similar for both enzymes. The results indicate that these 2 closely related serine proteases differ little in the structural arrangement and accessibility of the anionic pocket at which these inhibitors bind. The large differences observed with respect to substrate binding activity probably reflect substantial structural differences between the 2 enzymes at secondary sites adjacent to the primary anionic site.