Allosteric regulation provides a molecular mechanism for preferential utilization of the fully assembled dolichol-linked oligosaccharide by the yeastoligosaccharyltransferase

The oligosaccharyltransferase (OST) preferentially utilizes the fully assem bled dolichol-linked oligosaccharide Glc(3)Man(9)GlcNAc(2)-PP-Dol as the do nor for N-linked glycosylation of asparagine residues in N-X-T/S consensus sites in newly synthesized proteins. A wide variety of assembly intermediat es (Glc(0-2)Man(0-9)GlcNAc(2)-PP-Dol) can serve as the donor substrate for N-linked glycosylation of peptide acceptor substrates in vitro or of nascen t glycoproteins in mutant cells that are defective in donor substrate assem bly. A kinetic mechanism that can account for the selection of the fully as sembled donor substrate from a complex mixture of dolichol-linked oligosacc harides (OS-PP-Dol) has not been elucidated. Here, the steady-state kinetic properties of the OST were reinvestigated using a proteoliposome assay sys tem consisting of the purified yeast enzyme, near-homogeneous preparations of a dolichol-linked oligosaccharide (Glc(3)Man(9)GlcNAc(2)-PP-Dol or Man(9 )GlcNAc(2)-PP-Dol) and an I-125-labeled tripeptide as the acceptor substrat e. The K-m of the OST for the acceptor tripeptide was only slightly enhance d when GlC(3)Man(9)GlcNAc(2)-PP-Dol was the donor substrate relative to whe n Man(9)GlcNAc(2)-PP-Dol was the donor substrate. Evaluation of the kinetic data for both donor substrates showed deviations from typical Michaelis-Me nten kinetics. Sigmoidal saturation curves, Lineweaver-Burk plots with upwa rd curvature, and apparent Hill coefficients of about 1.4 suggested a subst rate activation mechanism involving distinct regulatory (activator) and cat alytic binding sites for OS-PP-Dol. Results of competition experiments usin g either oligosaccharide donor as an alternative substrate were also consis tent with this hypothesis. We propose that binding of either donor substrat e to the activator site substantially enhances Glc(3)Man(9)GlcNAc(2)-PP-Dol occupancy of the enzyme catalytic site via allosteric activation.