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 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.
Howard
S
S
Protein Engineering Network of Centres of Excellence of Canada, Vancouver, British Columbia.
Withers
S G
SG
D000345:Affinity Labels / Q000378:metabolism*; D002384:Catalysis; D002853:Chromatography, Liquid; D010450:Endopeptidases / Q000378:metabolism; D005960:Glucosides / Q000378:metabolism; D006027:Glycosides / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007659:Ketones / Q000378:metabolism*; D007700:Kinetics; D008362:Mannosides / Q000378:metabolism; D013058:Mass Spectrometry; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D000520:alpha-Glucosidases / Q000378:metabolism*
D000345:Affinity Labels; D005960:Glucosides; D006027:Glycosides; D007659:Ketones; D008362:Mannosides; D000520:alpha-Glucosidases; D010450:Endopeptidases