TAILIEUCHUNG - Báo cáo khoa học: The role of residues R97 and Y331 in modulating the pH optimum of an insect b-glycosidase of family 1

The activity of the digestiveb-glycosidase fromSpodoptera frugiperda (Sfbgly50, pH optimum ) depends on E399 (pKa¼; catalytic nucleophile) and E187 (pKa¼; 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 bymethionine or lysine increased the E399 pKa by or units, respectively, shifting the pH optima of these mutants to . | Eur. J. Biochem. 270 4866-4875 2003 FEBS 2003 doi The role of residues R97 and Y331 in modulating the pH optimum of an insect b-glycosidase of family 1 Sandro R. Marana Lucio M. F. Mendonca Eduardo H. P. Andrade Walter R. Terra and Clelia Ferreira Departamento de Bioquimica Instituto de Quimica Universidade de Scio Paulo Brazil The activity of the digestive b-glycosidase from Spodoptera frugiperda Sfbgly50 pH optimum depends on E399 pKa catalytic nucleophile and E187 pKa 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 pKa by or units respectively shifting the pH optima of these mutants to . The substitution of Y331 by phenylalanine increased the pKa of E399 and and units respectively and displaced the pH optimum to . From the observed ApKa it was calculated that R97 and Y331 contribute and 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 units and shifted the pH optimum to 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 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 pKa values pH optimum site-directed 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

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