| To elucidate the mechanism of the reaction catalyzed by yeast oligosaccharyl-transferase (OST, E.C. 2.4.1.119), the substrates have been truncated to meet the minimum requirements for the enzyme. Of two proposed enzyme-catalyzed activation processes, nucleophilic or electrophilic, possible nucleophilic activation can be investigated by isotopic labeling of the hydrogens in a tripeptide substrate. Using enzymatic and chemical syntheses, deuterium atoms have been incorporated stereospecifically into the {dollar}alpha{dollar} and {dollar}beta{dollar} positions of the asparagine in the tripeptide. (GlcNAc){dollar}sb2{dollar}-PP-Dolichol (lipid disaccharide, LDS) was chemically synthesized from chitin and polyisoprenols, giving greatly increased access to this substrate. The anticipated product of the enzyme-catalyzed reaction was also synthesized using a new convergent synthesis. To observe a possible isotopic exchange in the enzyme-catalyzed reaction, the deuterium-labeled tripeptides and chemically synthesized LDS were incubated in the presence of the P{dollar}sb{lcub}40{rcub}{dollar} yeast microsomes. The analysis of the biosynthesized glycopeptides by {dollar}sp1{dollar}H NMR showed the deuterium atoms were retained in the enzyme-catalyzed reaction, suggesting the enzyme does not catalyze the reaction by the two proposed mechanisms involving enol lactone and ketene intermediates. |
