Abstract
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 are likely involved in acid/base catalysis.
Highlights
The catalytic nucleophile, one as a general acid/base, and the third possibly affords additional stabilization of developing positive charge or modulates the ionization behavior of the other catalytic residues
In a second group of ␣-glycosyl hydrolases (Family 31), Asp-505 and Asp-1394 have been identified as active site residues in each of the two homologous active sites in the sucraseisomaltase complex by affinity labeling with conduritol B epoxide and have been suggested to be the catalytic nucleophiles [10]
The asparagine mutant of the equivalent Asp-518 in human lysosomal ␣-glucosidase exhibits an estimated 6% of wildtype activity, indicating that this residue likely plays an important role in catalysis [11], this 16-fold reduction in activity is much less than the 105-fold reduction observed upon analysis of nucleophile mutants in mechanistically similar -glycosidases [12,13,14], leaving some doubt concerning the role of Asp-518
Summary
The catalytic nucleophile, one as a general acid/base, and the third possibly affords additional stabilization of developing positive charge or modulates the ionization behavior of the other catalytic residues. The catalytic nucleophiles in several -glycosidases have been identified by trapping of these intermediates, followed by proteolytic digestion of the enzyme and identification of the labeled peptide(s) by electrospray mass spectrometry [13, 18, 19].
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Published Version
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