Effect of water miscible organic solvents on kinetics of a thermostable beta-glycosidase

A thermostable beta-D-glycosidase from Sulfolobus solfataricus was used to determine the effect of water-miscible solvents on enzyme activity and kine tics. Various o- and p-nitrophenvl glycosides were used as representative s ubstrates for hydrolysis. Initial enzyme activity was enhanced in the prese nce of methyl acetate, butan-2-one and acetonitrile. The activation by solv ent was found to be transitory, 1,4-Dioxane, formamide and tetrahydrofuran were inhibitory at all concentrations with all substrates used. Kinetic stu dies in the presence of solvents suggested that K-M values for all substrat es were unaffected by methyl acetate, but increased or decreased in the pre sence of 1.4-dioxane or tetrahydrofuran, in a substrate dependent manner. S tudies using an competitive inhibitor supported these results. Methyl aceta te did not change competitive-type inhibition by D-gluconic acid lactone, w hile in the presence of 1,4-dioxane altered the inhibition kinetics to mixe d-type, Tetrahydrofuran affected the mode of inhibition but not in a identi fiable manner. These results suggest that both 1,4-dioxane and tetrahydrofu ran may have a direct influence on the active site of the catalyst.