The kinetic and structural characterization of the reaction of nafamostat with bovine pancreatic trypsin.

Nafamostat mesilate (FUT-175), a synthetic serine protease inhibitor, is active against a number of the serine proteases involved in coagulation. This has been proposed as the basis of its anticoagulant activity. We investigated the reaction of Nafamostat with bovine pancreatic trypsin as a model system. It was shown to act as a time-dependent competitive inhibitor, and the inhibition constants for the binding of Nafamostat to trypsin (i.e., Ki) and the overall inhibition constants (i.e., Ki*) were calculated to be 11.5 microM and 0.4+/-0.14 nM, respectively. The second-order rate constant for the reaction was 4.5+/-0.19x10(5) M(-1)s(-1), and the product released following the acylation step, 6-amidino2-naphthol, showed mixed-type inhibition. The competitive (Kic) and uncompetitive (Kiu) inhibition constants were 14.7 microM and 19.5 microM, respectively. Formation of the acyl-enzyme intermediate was dissected into at least two steps, with rates of 0.9 s(-1) and 195 s(-1). The deacylation step was relatively much slower (3.2+/-0.19x10(-5) s(-1), enabling the mass spectroscopic analysis of the acyl-enzyme intermediate, which confirmed the covalent attachment of 4-guanidinobenzoic acid to trypsin. The product of the deacylation step, 4-guanidinobenzoic acid, showed no inhibition up to a concentration of 200 microM. These data strongly suggest that while Nafamostat is a potent inhibitor of trypsin, it is actually an extremely poor substrate, and that apparent inhibition is due to the competitive formation of a very stable acyl-enzyme intermediate, analogous to some other active site titrants.