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

Jiunly Chir, Stephen Withers, Chin Feng Wan, Yaw-Kuen Li*

*Corresponding author for this work

Research output: Contribution to journalArticle

31 Scopus citations

Abstract

β-Glucosidase from Flavobacterium meningosepticum (Fbg1) 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 N-bromoacetyl-β-D-glucosylamine (NBGN), which stoichiometrically label the nucleophile and the acid/base catalyst of Fbg1, respectively. Pseudo-first-order inactivation rate constants (ki) of 0.25 ± 0.01 and 0.05 ± 0.01 min-1 and dissociation constants (Ki) 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 Fbg1. 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.

Original languageEnglish
Pages (from-to)857-863
Number of pages7
JournalBiochemical Journal
Volume365
Issue number3
DOIs
StatePublished - 1 Aug 2002

Keywords

  • Electrospray ionization
  • General acid/base
  • Nucleophile
  • Peptide mapping

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