title: The kinetic and structural characterization of the reaction of nafamostat with bovine pancreatic trypsin.
abstract: 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.
authors.affiliation: Peptide Therapeutics, Cambridge, UK. manoj.ramjee@peptide.co.uk
authors.forename: M K
authors.initials: MK
authors.lastname: Ramjee
authors.forename: I M
authors.initials: IM
authors.lastname: Henderson
authors.forename: S B
authors.initials: SB
authors.lastname: McLoughlin
authors.forename: A
authors.initials: A
authors.lastname: Padova
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000737:chemistry / Q000378:metabolism; D001550:Benzamidines; D001565:Benzoates / Q000737:chemistry; D001665:Binding Sites; D002417:Cattle; D006146:Guanidines / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry; D008956:Models, Chemical; D015394:Molecular Structure; D009284:Naphthols / Q000737:chemistry; D010179:Pancreas / Q000201:enzymology; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000378:metabolism; D013057:Spectrum Analysis; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism*
chemical_list: D000925:Anticoagulants; D001550:Benzamidines; D001565:Benzoates; D006146:Guanidines; D009284:Naphthols; D015842:Serine Proteinase Inhibitors; C023856:4-guanidinobenzoate; C058874:6-amidino-2-naphthol; D014357:Trypsin; C032855:nafamostat