Single mutations outside the active site affect the substrate specificity in a β-glycosidase.

A library of random mutants of the β-glycosidase Sfβgly was screened for mutations that affect its specificity for the substrate glycone (β-d-fucoside versus β-d-glucoside). Among mutations selected (T35A, R189G, Y345C, P348L, S358F, S378G, N400D, S424F, F460L, and R474H), eight occurred in the C-terminal half of Sfβgly and only two were at the active site (R189G and N400D). Tryptophan fluorescence spectra and thermal inactivation showed that the selected mutants and wild-type Sfβgly are similarly folded. Enzyme kinetics confirmed that these mutations resulted in broadening or narrowing of the preference for the substrate glycone. Structural modeling and interaction maps revealed contact pathways that connect the sites of the selected mutations through up to three interactions to the active site residues E399, W444, and E187, which are involved in substrate binding and catalysis. Interestingly, independently selected mutations (Y345C, P348L, and R189G; S424F and N400D) were placed on the same contact pathway. Moreover, (k(cat)/K(m) fucoside)/(k(cat)/K(m) glucoside) ratios showed that mutations at intermediate residues of the same contact pathway often had similar effects on substrate specificity. Finally mutations in the same contact pathway caused similar structural disturbance as evidenced by acrylamide quenching of the Sfβgly fluorescence. Based on these data, it is proposed that the effects of the selected mutations were propagated into the active site through groups of interacting residues (contact pathways) changing the Sfβgly substrate specificity.