|Title||Labeling and identification of the postulated acid/base catalyst in the alpha-glucosidase from Saccharomyces cerevisiae using a novel bromoketone C-glycoside|
|Publication Type||Journal Article|
|Year of Publication||1998|
|Authors||Howard, S, Withers, SG|
|Date Published||MAR 17|
alpha-Glucosidase from Saccharomyces cerevisiae is a member of a sequence-related family of alpha-glycosidases (family 13) that includes digestive alpha-amylases and commercially important cyclodextrin glucanotransferases. These enzymes catalyze the hydrolysis of alpha-linked oligosaccharides by a two-step mechanism involving a glycosyl-enzyme intermediate. A novel bromoketone C-glycoside inactivator, 1'-bromo-3'-(alpha-D-mannopyranosyl)-2'-propanone, has been synthesized and used to label the putative acid/base catalyst (Glu-276) of yeast alpha-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 alpha-glucosidase and, thus, provide further evidence for this residue playing an important role in catalysis.