Active site titration of bovine β-trypsin by Nα-(N,N-dimethylcarbamoyl)-α-aza-lysine p-nitrophenyl ester: kinetic and crystallographic analysis

AbstractKinetics of bovine β-trypsin (trypsin) with the Nα-(N,N-dimethylcarbamoyl)-α-aza-lysine p-nitrophenyl ester (Dmc-azaLys-ONp) was obtained at pH 6.2 and 21.0°C. Dmc-azaLys-ONp shows the characteristics of an optimal active site titrant in that it (i) gives titrations in a short time, (ii) is a stable and soluble compound with a stoichiometric reaction that is easily and directly detectable, and (iii) allows titrations over a wide range of enzyme concentration. Moreover, the three-dimensional structure of the trypsin · Nα-(N-N-dimethylcarbamoyl)-α-aza-lysine acyl · enzyme adduct has been solved by X-ray crystallography at 2.0 Å resolution (R = 0.145). The Dmc-azaLys moiety of the active site titrant is sited in the serine proteinase reaction center, and is covalently linked to the OG atom of the Ser195 catalytic residue