The Trypsin-catalyzed Hydrolysis of Nα-Benzyloxycarbonyl-l-Lysine p-Nitrophenyl Ester in Dimethylsulfoxide at Sub-Zero Temperatures

Abstract

Abstract The effect of sub-zero temperatures and aqueous dimethylsulfoxide solutions on the trypsin-catalyzed hydrolysis of Nα-benzyloxycarbonyl-l-lysine p-nitrophenyl ester has been investigated. With increasing dimethylsulfoxide concentration at 0°, kcat decreases in proportion to the decreased water concentration; however, Km increases by 2 orders of magnitude. The effect on Km can be accounted for by a combination of dielectric and competitive inhibition effects. The Arrhenius plot for the deacylation reaction in 65 % aqueous dimethylsulfoxide is linear over the range 0 to -45° and extrapolates to a value of kcat at 25° in excellent agreement with that obtained in the absence of the organic solvent. At -45° and below, turnover occurred very slowly, but acylation was quite rapid. The effect of dimethylsulfoxide concentration and sub-zero temperatures on the ultraviolet spectral properties of the enzyme showed no evidence of any structural changes. All of the experimental observations were consistent with the conclusion that 65 % aqueous dimethylsulfoxide and sub-zero temperatures have no significant effect on the pathway of trypsin-catalyzed reactions. Preliminary results using Nα-acetyl-l-lysine methyl ester indicated that detectable perturbations in the environment of tyrosine and tryptophan residues in trypsin occur during catalysis.