Effects of methanol cryosolvents on the structural and catalytic properties of bovine trypsin.

The effect of aqueous methanol cryosolvents on the catalytic and structural properties of bovine trypsin has been investigated. The low freezing points and low viscosities of methanol-based cryosolvents are desirable for a variety of cryoenzymological experiments. Increasing concentrations of methanol caused increases in the values of kcat and Km for the hydrolysis of N alpha-benzyloxycarbonyl-L-lysine p-nitrophenyl ester at 0 degrees C and a small increase in Ki for inhibition by benzamidine. Based on product analysis the increase in kcat with increasing methanol concentration at pH* 4.0 and 6.5 can be completely accounted for by nucleophilic competition of methanol for the acyl enzyme intermediate. This observation indicates that deacylation is the rate-limiting step under these conditions. The effect of increasing methanol concentration on kcat/Km for the above ester substrate and N alpha-benzoyl-L-arginine p-nitroanilide was similar. Incubation experiments indicated that trypsin was quite stable in 70% methanol at 0 degrees C and below. The Arrhenius plot for the catalytic reaction in 70% methanol was linear over the 0 to -40 degrees C range, indicating no change in rate-determining step nor temperature-induced structural perturbation. No evidence for structural effects induced by methanol or temperature were detected by monitoring the intrinsic fluorescence and absorbance. We conclude that aqueous methanol cryosolvents are satisfactory for cryosolvent studies of trypsin.