Oligosaccharyltransferase: synthesis and use of deuterium-labeled peptide substrates as mechanistic probes.
Chemically synthesized peptide and lipid disaccharide substrates have been used to investigate two possible mechanisms for enzyme-catalyzed N-glycosylation. Using microsomal oligosaccharyltransferase isolated from yeast, the fate of the deuterium in three stereospecifically deuterated peptides has been investigated. In all three cases, the deuterium present in the peptide substrate was retained in the glycopeptide product, as shown clearly by 1H NMR spectral comparisons. The lack of deuterium wash-out during catalysis provides strong evidence against either enol lactone or ketene formation as an intermediate in this reaction.
Lee
J
J
Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor 48109.
Coward
J K
JK
D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D003903:Deuterium; D006020:Glycopeptides / Q000096:biosynthesis*; D006031:Glycosylation; D006602:Hexosyltransferases; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008565:Membrane Proteins; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity; D014166:Transferases / Q000378:metabolism*
D006020:Glycopeptides; D008565:Membrane Proteins; D009842:Oligopeptides; D003903:Deuterium; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase