title: The role of the hydroxy amino acid in the triplet sequence Asn-Xaa-Thr(Ser) for the N-glycosylation step during glycoprotein biosynthesis.
abstract: The catalytical role of the hydroxy amino acid in the "marker sequence" Asn-Xaa-Thr(Ser) for the N-glycosylation step of glycoprotein formation was investigated by using a series of hexapeptides derived from Tyr-Asn-Gly-Xaa-Ser-Val by substituting threonine, serine, cysteine, valine and O-methylthreonine respectively for Xaa. The results, which were obtained with calf liver microsomal fractions as enzyme source and dolichyl diphosphate di-N-acetyl [14C] chitobiose as glycosyl donor showed that the threonine-, serine- and cysteine-containing derivatives could be glycosylated, although at very different rates, whereas the valine and O-methylthreonine analogues did not work as glycosyl acceptors. Replacement of threonine by serine resulted in a 4-fold decrease in Vmax, and about a 10-fold increase in Km for glycosyl transfer. Replacement of serine by cysteine again decreased acceptor activity 2-3-fold. The various results, taken together, indicate an absolute requirement for a hydrogen-bond-donor function in the side chain of the hydroxy amino acid of the "marker sequence" and furthermore, point to a considerable influence of the structure of this amino acid on binding as well as on the glycosyl transfer itself. In order to explain the observed differences in the glycosyl-transfer rates, a model is proposed with a hydrogen-bond interaction between the amide of asparagine as the hydrogen-bond donor and the oxygen of the hydroxy group of the hydroxy amino acid as the hydrogen-bond acceptor. The participation of the hydroxy group in the catalytic mechanism of glycosyl transfer in the kind of proton-relay system is discussed.
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D006023:Glycoproteins / Q000096:biosynthesis*; D006602:Hexosyltransferases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology; D008954:Models, Biological; D010455:Peptides / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D006023:Glycoproteins; D010455:Peptides; D006602:Hexosyltransferases; C018660:asparagine-N-glycosyltransferase