Unequivocal identification of Asp-214 as the catalytic nucleophile of Saccharomyces cerevisiae alpha-glucosidase using 5-fluoro glycosyl fluorides.
McCarter J.D. | Withers S.G.
Yeast alpha-glucosidase is a member of a sequence-related family of alpha-glycosidases (Family 13) that includes important digestive alpha-amylases and alpha-glucosidases. These enzymes catalyze the hydrolysis of alpha-linked oligosaccharides by a two-step mechanism involving a glycosyl-enzyme intermediate. This intermediate can be trapped by use of 5-fluoro-alpha-D-glucosyl fluoride or 5-fluoro-beta-L-idosyl fluoride, members of a new class of mechanism-based glycosidase inactivators. Both of these trapped 5-fluoro glycosyl enzyme intermediates are catalytically competent, turning over when freed of excess inactivator and releasing free enzyme. Two glycosylated peptides in proteolytic digests of these trapped glycosyl enzyme intermediates were identified by use of neutral loss scans on an electrospray ionization triple quadrupole mass spectrometer. Further tandem mass spectrometric analysis in daughter ion scan mode allowed identification of Asp-214 as the catalytic nucleophile in yeast alpha-glucosidase, and this identification was confirmed by aminolysis of the labeled peptide and high resolution mass spectrometry. This residue is one of three active site carboxylates that are completely conserved in this family, thus confirming the role of Asp-214 and the equivalent residues in other family members as the catalytic nucleophile. The other two conserved carboxylates likely involved in acid/base catalysis.
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