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

A library of random mutants of the ß-glycosidase Sfßgly was screened for mutations that affect its specificity for the substrate glycone (ß-d-fucoside versus ß-d-glucoside). Among mutations selected (T35A, R189G, Y345C, P348L, S358F, S378G, N400D, S424F, F460L, and R474H), eight occurred in the C-terminal half of Sfßgly 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 Sfßgly 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 Sfßgly 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 Sfßgly substrate specificity.

The role in the substrate specificity and catalysis of residues forming the substrate aglycone-binding site of a beta-glycosidase.

The relative contributions to the specificity and catalysis of aglycone, of residues E190, E194, K201 and M453 that form the aglycone-binding site of a beta-glycosidase from Spodoptera frugiperda (EC 3.2.1.21), were investigated through site-directed mutagenesis and enzyme kinetic experiments. The results showed that E190 favors the binding of the initial portion of alkyl-type aglycones (up to the sixth methylene group) and also the first glucose unit of oligosaccharidic aglycones, whereas a balance between interactions with E194 and K201 determines the preference for glucose units versus alkyl moieties. E194 favors the binding of alkyl moieties, whereas K201 is more relevant for the binding of glucose units, in spite of its favorable interaction with alkyl moieties. The three residues E190, E194 and K201 reduce the affinity for phenyl moieties. In addition, M453 favors the binding of the second glucose unit of oligosaccharidic aglycones and also of the initial portion of alkyl-type aglycones. None of the residues investigated interacted with the terminal portion of alkyl-type aglycones. It was also demonstrated that E190, E194, K201 and M453 similarly contribute to stabilize ES(double dagger). Their interactions with aglycone are individually weaker than those formed by residues interacting with glycone, but their joint catalytic effects are similar. Finally, these interactions with aglycone do not influence glycone binding.

The role of residues R97 and Y331 in modulating the pH optimum of an insect beta-glycosidase of family 1.

The activity of the digestive beta-glycosidase from Spodoptera frugiperda (Sfbetagly50, pH optimum 6.2) depends on E399 (pKa = 4.9; catalytic nucleophile) and E187 (pKa = 7.5; catalytic proton donor). Homology modelling of the Sfbetagly50 active site confirms that R97 and Y331 form hydrogen bonds with E399. Site-directed mutagenesis showed that the substitution of R97 by methionine or lysine increased the E399 pKa by 0.6 or 0.8 units, respectively, shifting the pH optima of these mutants to 6.5. The substitution of Y331 by phenylalanine increased the pKa of E399 and E187 by 0.7 and 1.6 units, respectively, and displaced the pH optimum to 7.0. From the observed deltapKa it was calculated that R97 and Y331 contribute 3.4 and 4.0 kJ.mol(-1), respectively, to stabilization of the charged E399, thus enabling it to be the catalytic nucleophile. The substitution of E187 by D decreased the pKa of residue 187 by 0.5 units and shifted the pH optimum to 5.8, suggesting that an electrostatic repulsion between the deprotonated E399 and E187 may increase the pKa of E187, which then becomes the catalytic proton donor. In short the data showed that a network of noncovalent interactions among R97, Y331, E399 and E187 controls the Sfbetagly50 pH optimum. As those residues are conserved among the family 1 beta-glycosidases, it is proposed here that similar interactions modulate the pH optimum of all family 1 beta-glycosidases.