INHIBITION OF BOVINE BETA-TRYPSIN BY THE ACTIVE-SITE TITRANT (N)UNDER-BAR-DIMETHYLCARBAMOYL)-ALPHA-AZAORNITHINE (P)UNDER-BAR-NITROPHENYL ESTER - A KINETIC AND X-RAY CRYSTALLOGRAPHIC STUDY

Kinetics of the bovine beta-trypsin (trypsin) reaction with the active site titrant (N) under bar(alpha)((N) under bar,(N) under bar-dimethy lcarbamoyl)-alpha-azaornithine (p) under bar-nitrophenyl ester (Dmc-az aOrn-ONp) was obtained at pH 6.2 and 21.0 degrees C. The results are c onsistent with the minimum three-step catalytic mechanism of serine pr oteinases involving a stable acyl . enzyme adduct. Dmc-azaOrn-ONp bind s stoichiometrically to trypsin and allows the reliable determination of the active enzyme concentration between 1.0x10(-6) M and 3.0x10(-4) M. The three-dimensional structure of the trypsin . Dmc-azaOrn acyl . enzyme adduct has been solved by X-ray crystallography at 1.8 Angstro m resolution ((R) under bar = 0.153). The Dmc-azaOrn moiety of the act ive site titrant is accommodated in the serine proteinase active cente r, occupying the S-1 specificity subsite, and is covalently linked to the OG atom of the Ser195 catalytic residue. (C) 1995 Academic Press, Inc.