The activity of the digestive b-glycosidase from Spodoptera frugiperda (Sfbgly50, pH optimum 6.2) depends on E399 (pK a ¼ 4.9; catalytic nucleophile) and E187 (pK a ¼ 7.5; catalytic proton donor). Homology modelling of the Sfbgly50 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 pK a 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 pK a of E399 and E187 by 0.7 and 1.6 units, respectively, and displaced the pH optimum to 7.0. From the observed DpK a it was calculated that R97 and Y331 contribute 3.4 and 4.0 kJAEmol )1 , respectively, to stabilization of the charged E399, thus enabling it to be the catalytic nucleophile. The substitution of E187 by D decreased the pK a 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 pK a 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 Sfbgly50 pH optimum. As those residues are conserved among the family 1 b-glycosidases, it is proposed here that similar interactions modulate the pH optimum of all family 1 b-glycosidases.Keywords: b-glycosidase; pK a values; pH optimum; sitedirected mutagenesis; Spodoptera frugiperda.The b-glycosidases from glycoside hydrolase family 1 are enzymes that remove monosaccharides from the nonreducing end of di-and/or oligosaccharides. According to the CAZy website this family comprises 422 sequenced b-glycosidases, of which the tertiary structure of 12 has been determined. Together with families 2, 10,17,26, 30, 35, 39, 42, 51, 53, 59, 72, 79 and 86 family 1 forms clan A, a group of families that shares structural and catalytic similarities [1]. All b-glycosidases of family 1 present the same tertiary structure [the (b/a) 8 barrel], they are configuration-retaining glycosidases and their catalytic activity depends on two glutamic acid residues, one positioned after b strand 4 and the other after b strand 7 [1]. These glutamic acids are very close inside the active site (about 4.5 Å apart) [2], and during the reaction the first glutamic acid acts as proton donor, and the second acts as a nucleophile. The catalytic nucleophile pK a is around 5.0 and the catalytic proton donor pK a is around 7.0 [3][4][5][6][7].A plot of b-glycosidase activity vs. pH presents a bell shape, indicating that in the pH optimum the catalytic nucleophile is deprotonated and the catalytic proton donor is protonated. Hence the branch of the curve below the pH optimum is determined mainly by the ionization of the catalytic nucleophile, whereas the catalytic ionization of the proton donor determines the branch above the pH optimum.As the b-glycosidase activity depends on the finely tuned ionization of the catalytic nucleophile and proton ...

The specificity of the Spodoptera frugiperda digestive b-glycosidase (Sfbgly50) for fucosides, glucosides and galactosides is determined by noncovalent interactions of glycone 6-OH and glycone 4-OH with the active-site residues Q39 and E451. Site-directed mutagenesis and enzyme steadystate kinetics were described, showing that replacement of E451 with glutamine increased the preference of Sfbgly50 for glucosides in comparison to galactosides, whereas replacing E451 with serine had the opposite effect. In contrast, the replacement of E451 with aspartate did not change Sfbgly50 specificity. The energy of the interactions formed by these different residues with the axial and equatorial glycone 4-OH were also measured, showing that the increase in preference for galactosides resulted from a larger energy decrease in the interaction with equatorial 4-OH than with axial 4-OH (22.6 vs. 13.9 kJAEmol ). The introduction of glutamine at position 451 or of asparagine at position 39 increased the preference of Sfbgly50 for fucosides in comparison to galactosides, whereas the presence of aspartate or serine at position 451 had less effect on this preference. The hydrolysis of fucosides was favored because glutamine at position 451 increased a steric hindrance with 6-OH of 7.1 kJAEmol )1 and asparagine at position 39 disrupted a favorable interaction with this same hydroxyl. In conclusion, it is proposed that the specificity of new b-glycosidase mutants can be predicted by combining and adding energy of the enzyme-substrate interactions evaluated in the present study.Keywords: b-glycosidase; bioenergetics analysis; enzyme specificity; glycoside hydrolase; site-directed mutagenesis.b-glycosidases from glycoside hydrolase family 1 are enzymes that remove monosaccharides from the nonreducing end of di-and/or oligosaccharides. The nonreducing monosaccharide residue binds at the glycone subsite (subsite )1), whereas the remaining part of the substrate is accommodated by the aglycone subsite 2 , which may actually be composed of several subsites (+1, +2, +3, etc.). According to the CAZy database, family 1 currently comprises 427 b-glycosidases, with 3D structural data being available for 12 [1]. All family 1 b-glycosidases share the same tertiary structure [(b/a) 8 barrel]; they are configuration-retaining glycosidases, the catalytic activity of which depends on two glutamic acid residues, one positioned after the b strand 4 (catalytic proton donor) and the other after the b strand 7 (catalytic nucleophile). Family 1 comprises enzymes with 14 different EC numbers 3 , catalyzing hydrolysis of substrates presenting a variety of glycones (monosaccharides such as glucose, galactose, fucose, mannose, 6-phosphoglucose and 6-phosphogalactose) and aglycones (monosaccharides, oligosaccharides, alkyl and aryl moieties) [1]. This broad substrate specificity makes family 1 an interesting model for using to study the molecular basis of the enzymatic specificity.Having a better understanding of the molecular basis of b-glycosidase specificity wo...