Article ID Journal Published Year Pages File Type
2049929 FEBS Letters 2007 6 Pages PDF
Abstract

The mechanism-based inhibitor 2,4-dinitrophenyl 2-deoxy-2-fluoro-β-d-galactopyranoside (DNP2FGal) was used to inactivate the Family 42 β-galactosidase (YesZ) from Bacillus subtilis via the trapping of a glycosyl-enzyme intermediate, thereby tagging the catalytic nucleophile in the active site. Proteolytic digestion of the inactivated enzyme and of a control sample of unlabeled enzyme, followed by comparative high-performance liquid chromatography and mass spectrometric analysis identified a labelled peptide of the sequence ETSPSYAASL. These data, combined with sequence alignments of this region with representative members of Family 42, unequivocally identify the catalytic nucleophile in this enzyme as Glu-295, thereby providing the first direct experimental proof of the identity of this residue within Family 42.

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