N-Glycosylation of yeast proteins. Characterization of the solubilized oligosaccharyl transferase.
The enzyme transferring the oligosaccharide from DolPP-(GlcNAc)2(Glc)3 to asparagine residues of glycoproteins has been solubilized from yeast membranes by extraction with detergents. Enzyme activity was tested by measuring transfer of the glycosyl moiety from DolPP-[14C]saccharides to the hexapeptide Tyr-Asn-Leu-Thr-Ser-Val. The rate of transfer was linear for 20 min, with about 40% of dolichyl-diphosphate-bound radioactivity transferred to the peptide. The solubilized enzyme has been characterized as follows: 1. The enzyme is most efficiently solubilized (60% of the membrane-associated activity) by 0.5% Nonidet P40 at a protein/detergent ratio of 2. Octylglucoside solubilizes one third of the activity, but strongly inhibits the reaction if present in the test at a concentration of 1%. 2. Divalent cations are absolutely required. 1 mM Mn2+ is optimal; Mg2+ at a concentration of 10mM yields only one third the activity observed with Mn2+. 3. The enzyme transfers besides dolichyl-diphosphate-bound (GlcNAc)2(Man)9(Glc)3 also (GlcNAc)2(Man)1 and (GlcNAc)2; the rate decreases in this order. No transfer is observed from DolPP-(GlcNAc)2(Man)9 and from DolPP-GlcNAc. 4. The Km value for DolPP-(GlcNAc)2(Man)9(Glc)3 of 0.5 microM does not differ significantly from that for DolPP-(GlcNAc)2 of 1.2 microM. A broad pH-optimum for the reaction with both substrates was found between 6.5 and 7.7. 5. However, a clear difference in Km values for the hexapeptide was observed with difference dolichol-linked sugar derivatives. With DolPP-(GlcNAc)2 a peptide concentration of 0.6 mM resulted in half-maximal transfer rate, whereas 0.05 mM peptide were sufficient with DolPP-(GlcNAc)2(Man)9(Glc)3 as donor.
D002413:Cations, Divalent; D005656:Fungal Proteins / Q000378:metabolism*; D006602:Hexosyltransferases; D007700:Kinetics; D008565:Membrane Proteins; D011104:Polyisoprenyl Phosphate Oligosaccharides; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012995:Solubility; D013379:Substrate Specificity; D014166:Transferases / Q000302:isolation & purification / Q000378:metabolism*
D002413:Cations, Divalent; D005656:Fungal Proteins; D008565:Membrane Proteins; D011104:Polyisoprenyl Phosphate Oligosaccharides; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase