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 a lpha G1cF) was used to trap a glycosyl-enzyme intermediate and identify the catalytic nucleophile at the active site of Aspergillus niger alpha -gluco sidase (Family 31). Incubation of the enzyme with 5FaG1cF, followed by pept ic 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 aspar tic acid residue (D). Comparison with the known protein sequence identified the labelled amino acid as Asp-224 of the P2 subunit.