UNEQUIVOCAL IDENTIFICATION OF ASP-214 AS THE CATALYTIC NUCLEOPHILE OFSACCHAROMYCES-CEREVISIAE ALPHA-GLUCOSIDASE USING 5-FLUORO GLYCOSYL FLUORIDES

Yeast alpha-glucosidase is a member of a sequence-related family of al pha-glycosidases (Family 13) that includes important digestive alpha-a mylases and alpha-glucosidases. These enzymes catalyze the hydrolysis of alpha-linked oligosaccharides by a two-step mechanism involving a g lycosyl-enzyme intermediate. This intermediate can be trapped by use o f 5-fluoro-alpha-D-glucosyl fluoride or 5-fluoro-beta-L-idosyl fluorid e, members of a new class of mechanism-based glycosidase inactivators. Both of these trapped 5-fluoro glycosyl enzyme intermediates are cata lytically competent, turning over when freed of excess inactivator and releasing free enzyme. Two glycosylated peptides in proteolytic diges ts of these trapped glycosyl enzyme intermediates were identified by u se of neutral loss scans on an electrospray ionization triple quadrupo le mass spectrometer. Further tandem mass spectrometric analysis in da ughter ion scan mode allowed identification of Asp-214 as the catalyti c nucleophile in yeast alpha-glucosidase, and this identification was confirmed by aminolysis of the labeled peptide and high resolution mas s 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 are likel y involved in acid/base catalysis.