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-a lpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester (Dmc- azaLys-ONp) catalyzed by bovine beta-trypsin, bovine alpha-thrombin, h uman alpha-thrombin, human Lys77-plasmin, human urinary kallikrein, th e M(r) 33,000 and M(r) 54,000 species of human urokinase, as well as b y porcine pancreatic beta-kallikrein-A and B have been obtained betwee n pH 6.0 and 8.0, at 21.0 degrees C. Moreover, the three dimensional s tructure of the human alpha-thrombin-(hirugen). Dmc-azaLys acyl . enzy me 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 b ovine beta-trypsin . Dmc-azaLys acyl . enzyme structure, a second, not covalently bound, inhibitor molecule, partly shielded by the 60-inser tion loop of human alpha-thrombin, is contacting the enzyme ''aryl-bin ding site''. (C) 1996 Academic Press Limited