Labeling and Identification of the Postulated Acid/Base Catalyst in the α-Glucosidase from Saccharomyces cerevisiae Using a Novel Bromoketone C-Glycoside

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Abstract
α-Glucosidase from Saccharomyces cerevisiae is a member of a sequence-related family of α-glycosidases (family 13) that includes digestive α-amylases and commercially important cyclodextrin glucanotransferases. These enzymes catalyze the hydrolysis of α-linked oligosaccharides by a two-step mechanism involving a glycosyl−enzyme intermediate. A novel bromoketone C-glycoside inactivator, 1‘-bromo-3‘-(α-d-mannopyranosyl)-2‘-propanone, has been synthesized and used to label the putative acid/base catalyst (Glu-276) of yeast α-glucosidase. Electrospray ionization mass spectrometry was used to demonstrate stoichiometric labeling of the protein. The labeled residue was identified by comparative liquid chromatographic/mass spectrometric analysis of peptic digests of labeled and unlabeled enzyme samples, which confirmed the unique presence of two labeled peptides of m/z 745 and 694. Subsequent tandem mass spectrometric analysis in the daughter-ion scan mode showed the two peptides to have an overlapping sequence in which Glu-276 was the labeled residue. Together with active-site-directed protection against inactivation with deoxynojirimycin, these results prove that Glu-276 is located within the active site of yeast α-glucosidase and, thus, provide further evidence for this residue playing an important role in catalysis.