Identification of the two essential groups in the family 3 beta-glucosidase from Flavobacterium meningosepticum by labelling and tandem mass spectrometric analysis.
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.
Chir
Jiunly
J
Department of Applied Chemistry, National Chiao Tung University, 1001 Ta-Hseh Road, Hsin-Chu, Taiwan, 30050, R.O.C.
Withers
Stephen
S
Wan
Chin-Feng
CF
Li
Yaw-Kuen
YK
D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D005417:Flavobacterium / Q000737:chemistry / Q000201:enzymology*; D005960:Glucosides / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000378:metabolism; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D005960:Glucosides; D010455:Peptides; C018852:N-bromoacetylglucosamine; D001617:beta-Glucosidase; D000117:Acetylglucosamine