Identification of the two essential groups in the family 3 β-glucosidase from Flavobacterium meningosepticum by labelling and tandem mass spectrometric analysis

摘要

pβ-Glucosidase from iFlavobacterium meningosepticum/i (Fbgl) catalyses the hydrolysis of β-1,4-glucosidic bonds via a two-step double-displacement mechanism in which two amino acid residues act as nucleophile and acid/base catalyst. Definitive identification of these two residues is provided by the two active-site-directed inactivators, 2′,4′-dinitrophenyl-2-deoxy-2-fluoro-β-d-glucoside (2FDNPG) and iN/i-bromoacetyl-β-d-glucosylamine (NBGN), which stoichiometrically label the nucleophile and the acid/base catalyst of Fbgl, respectively. Pseudo-first-order inactivation rate constants (iksubi/sub/i) of 0.25±0.01 and 0.05±0.01minsup?1/sup and dissociation constants (iK/isubi/sub) of 90±15 and 4.4±0.2mM are determined for 2FDNPG and NBGN, respectively. Proteolytic digestion of the labelled proteins, followed by peptide mapping and tandem MS analysis identify Asp-247 and Glu-473 as the catalytic nucleophile and acid/base residues, respectively, of Fbgl. This study confirms that the catalytic nucleophile of family 3 glycohydrolase is conserved across sub-families. However, different sub-families may have unique general acid/base catalysts./p