Kinetics of bovine beta-trypsin (trypsin) with the N alpha-(N,N-dimethylcarbamoyl)-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 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.N alpha-(N,N-dimet hylcarbamoyl)-alpha-aza-lysine acyl.enzyme adduct has been solved by X-ray crystallography at 2.0 A 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.
Active site titration of bovine beta-trypsin by N alpha-(N,N-dimethylcarbamoyl)-alpha-aza-lysine p-nitrophenyl ester: kinetic and crystallographic analysis
BALLIANO, Gianni;MILLA, Paola;
1995-01-01
Abstract
Kinetics of bovine beta-trypsin (trypsin) with the N alpha-(N,N-dimethylcarbamoyl)-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 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.N alpha-(N,N-dimet hylcarbamoyl)-alpha-aza-lysine acyl.enzyme adduct has been solved by X-ray crystallography at 2.0 A 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.
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