LABELING AND IDENTIFICATION OF THE POSTULATED ACID BASE CATALYST IN THE ALPHA-GLUCOSIDASE FROM SACCHAROMYCES-CEREVISIAE USING A NOVEL BROMOKETONE C-GLYCOSIDE/

alpha-Glucosidase from Saccharomyces cerevisiae is a member of a seque nce-related family of alpha-glycosidases (family 13) that includes dig estive alpha-amylases and commercially important cyclodextrin glucanot ransferases. These enzymes catalyze the hydrolysis of alpha-linked oli gosaccharides by a two-step mechanism involving a glycosyl-enzyme inte rmediate. A novel bromoketone C-glycoside inactivator, 1'-bromo-3'-(al pha-D-mannopyranosyl)-2'-propanone, has been synthesized and used to l abel the putative acid/base catalyst (Glu-276) of yeast alpha-glucosid ase. Electrospray ionization mass spectrometry Was used to demonstrate stoichiometric labeling of the protein. The labeled residue was ident ified by comparative liquid chromatographic/mass spectrometric analysi s of peptic digests of labeled and unlabeled enzyme samples, which con firmed the unique presence of two labeled peptides of m/z 745 and 694. Subsequent tandem mass spectrometric analysis in the daughter-ion sca n mode showed the two peptides to have an overlapping sequence in whic h Glu-276 was the labeled residue. Together with active-site-directed protection against inactivation with deoxynojirimycin, these results p rove that Glu-276 is located within the active site of yeast alpha-glu cosidase and, thus, provide further evidence for this residue playing an important role in catalysis.