Identification of the catalytic nucleophile of the Family 31 alpha-glucosidase from Aspergillus niger via trapping of a 5-fluoroglycosyl-enzyme intermediate.

The mechanism-based reagent 5-fluoro-alpha-d-glucopyranosyl fluoride (5F alpha GlcF) was used to trap a glycosyl-enzyme intermediate and identify the catalytic nucleophile at the active site of Aspergillus niger alpha-glucosidase (Family 31). Incubation of the enzyme with 5F alpha GlcF, followed by peptic proteolysis and comparative liquid chromatography/MS mapping allowed the isolation of a labelled peptide. Fragmentation analysis of this peptide by tandem MS yielded the sequence WYDMSE, with the label located on the aspartic acid residue (D). Comparison with the known protein sequence identified the labelled amino acid as Asp-224 of the P2 subunit.