標題: Identification of the two essential groups in the family 3 beta-glucosidase from Flavobacterium meningosepticum by labelling and tandem mass spectrometric analysis
作者: Chir, J
Withers, S
Wan, CF
Li, YK
應用化學系
Department of Applied Chemistry
關鍵字: electrospray ionization;general acid/base;nucleophile;peptide mapping
公開日期: 1-Aug-2002
摘要: beta-Glucosidase from Flavobacterium meningosepticum (Fbgl) catalyses the hydrolysis of beta-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-beta-D-glucoside (2FDNPG) and N-bromoacetyl-beta-D-glucosylamine (NBGN), which stoichiometrically label the nucleophile and the acid/base catalyst of Fbgl, respectively. Pseudo-first-order inactivation rate constants (k(i)) of 0.25+/-0.01 and 0.05+/-0.01 min(-1) and dissociation constants (K-i) of 90+/-15 and 4.4+/-0.2 mM 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.
URI: http://dx.doi.org/10.1042/BJ20020186
http://hdl.handle.net/11536/28611
ISSN: 0264-6021
DOI: 10.1042/BJ20020186
期刊: BIOCHEMICAL JOURNAL
Volume: 365
Issue: 
起始頁: 857
結束頁: 863
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