Human alpha-thrombin inhibition by the active site titrant N-alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester: A comparative kinetic and X-ray crystallographic study

Kinetics for the hydrolysis of the chromogenic active site titrant N-alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp) catalyzed by bovine beta-trypsin, bovine alpha-thrombin, human alpha-thrombin, human Lys77-plasmin, human urinary kallikrein, the M(r) 33,000 and M(r) 54,000 species of human urokinase, as well as by porcine pancreatic beta-kallikrein-A and B have been obtained between pH 6.0 and 8.0, at 21.0 degrees C. Moreover, the three dimensional structure of the human alpha-thrombin-(hirugen). Dmc-azaLys acyl . enzyme complex has been analyzed and refined by X-ray crystallography at 2.0 Angstrom resolution (R-factor = 0.168). As observed for bovine beta-trypsin, the acylating inhibitor molecule is covalently bound to the Ser195 catalytic residue, filling the human alpha-thrombin S-1 primary specificity subsite with its lysyl side-group. However, the carbonyl group of the scissile human alpha-thrombin . Dmc-azalys acyl bond does not occupy properly the oxyanion binding hole. At variance from the bovine beta-trypsin . Dmc-azaLys acyl . enzyme structure, a second, not covalently bound, inhibitor molecule, partly shielded by the 60-insertion loop of human alpha-thrombin, is contacting the enzyme ''aryl-binding site''. (C) 1996 Academic Press Limited