ACTIVE-SITE TITRATION OF BOVINE BETA-TRYPSIN BY N-ALPHA-(N,N-DIMETHYLCARBAMOYL)-ALPHA-AZA-LYSINE P-NITROPHENYL ESTER - KINETIC AND CRYSTALLOGRAPHIC ANALYSIS

Kinetics of bovine beta-trypsin (trypsin) with the N-alpha-(N,N-dimeth ylcarbamoyl)-alpha-aza-lysine p-nitrophenyl ester (Dmc-azaLys-ONp) was obtained at pH 6.2 and 21.0 degrees C. Dmc-azaLys-ONp shows the chara cteristics of an optimal active site titrant in that it (i) gives titr ations in a short time, (ii) is a stable and soluble compound with a s toichiometric reaction that is easily and directly detectable, and (ii i) allows titrations over a wide range of enzyme concentration. Moreov er, the three-dimensional structure of the trypsin.N-alpha-(N,N-dimet hylcarbamoyl)-alpha-aza-lysine acyl enzyme adduct has been solved by X -ray crystallography at 2.0 Angstrom resolution (R = 0.145). The Dmc-a zaLys moiety of the active site titrant is sited in the serine protein ase reaction center, and is covalently linked to the OG atom of the Se r(195) catalytic residue.