Cryoenzymology of β-galactosidase: Effects of cryosolvents

Abstract

Experimental support for the use of fluid aqueous organic solvent systems and subzero temperatures in mechanistic studies of β-galactosidase is presented. The enzyme was stable and retained catalytic activity and structural integrity in 50% aqueous dimethyl sulfoxide and 60% aqueous methanol at 0°C; at lower temperatures higher concentrations of cosolvent may be successfully used. The effects of dimethyl sulfoxide on the catalytic and structural properties of the enzyme were investigated in detail. For the β-galactoside-catalyzed h ydrolysis ofo-nitrophenyl-β-D-galactoside the value ofk cat decreased in a linear manner with increasing cosolvent concentration, whereasK m increased exponentially. The decrease ink cat paralleled the decrease in water concentration, consistent with rate-limiting hydrolysis of a galactosylenzyme intermediate. The increase inK m is attributed to less favorable partitioning of the substrate to the active site in the cryosolvent compared to aqueous solution. ThepH*-rate profile for this reaction at 0°C in 50% dimethyl sulfoxide was similar to that in aqueous solution, withpK*1=5.8 andpK*2=8.0. Linear Arrhenius plots, with energies of activation of 13.9 and 16.0 kcal mol−1, respectively, were obtained for the β-galactosidase-catalyzed hydrolysis ofo-nitrophenyl- andp-nitrophenyl-β-D-galactosides in 50% dimethyl sulfoxide at temperatures to −57°C. Examination of the intrinsic fluorescence and ultraviolet spectra of the enzyme as a function of increasing cosolvent concentration showed no evidence for structural perturbation up to and including 50% dimethyl sulfoxide at 0°C. We conclude that these cryosolvent systems are suitable for mechanistic investigations of β-galactosidase, in particular for trapping intermediates at subzero temperatures.