Journal
GLYCOBIOLOGY
Volume 21, Issue 5, Pages 547-552Publisher
OXFORD UNIV PRESS INC
DOI: 10.1093/glycob/cwq190
Keywords
electrospray ionization mass spectrometry; glycosylation; glycosyl-enzyme intermediate; human ABO (H) blood group glycosyltransferases; retaining glycosyltransferase
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Funding
- Alberta Ingenuity Centre for Carbohydrate Science
- Natural Sciences and Engineering Research Council of Canada
- Danish Natural Sciences Council
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The enzymatic mechanism by which retaining glycosyltransferases (GTs) transfer monosaccharides with net retention of the anomeric configuration has, so far, resisted elucidation. Here, direct detection of covalent glycosyl-enzyme intermediates for mutants of two model retaining GTs, the human blood group synthesizing alpha-(1 -> 3)-N-acetylgalactosaminyl-transferase (GTA) and alpha-(1 -> 3)-galactosyltransferase (GTB) mutants, by mass spectrometry (MS) is reported. Incubation of mutants of GTA or GTB, in which the putative catalytic nucleophile Glu(303) was replaced with Cys (i.e. GTA(E303C) and GTB(E303C)), with their respective donor substrate results in a covalent intermediate. Tandem MS analysis using collision-induced dissociation confirmed Cys(303) as the site of glycosylation. Exposure of the glycosyl-enzyme intermediates to a disaccharide acceptor results in the formation of the corresponding enzymatic trisaccharide products. These findings suggest that the GTA(E303C) and GTB(E303C) mutants may operate by a double-displacement mechanism.
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