Regulation of the biosynthesis of N-acetylglucosaminylpyrophosphoryldolichol feedback and product inhibition

The assembly of the core oligosaccharide region of asparagine-linked glycop roteins proceeds by means of the dolichol pathway, The first step of this p athway, the reaction of dolichol phosphate with UDP-GlcNAc to formN-acetylg lucosaminylpyrophosphoryldolichol (GlcNAc-P-P-dolichol), is under investiga tion as a possible site of metabolic regulation, This report describes feed back inhibition of this reaction by the second intermediate of the pathway, N-acetylglucosaminyl-N-acetylglucosaminylpyrophosphoryldolichol (GlcNAc-Gl cNAc-P-P-dolichol), and product inhibition by GlcNAc-P-P-dolichol itself. T hese influences were revealed when the reactions mere carried out in the pr esence of showdomycin, a nucleoside antibiotic, present at concentrations t hat block the cte novo formation of GlcNAc-GlcNAc-P-P-dolichol but not that of GlcNAc-P-P-dolichol. The apparent Ki values for GlcNAc-P-P-dolichol and GlcNAc-GlcNAc-P-P-dolichol under basal conditions were 4.4 and 2.8 mu m, r espectively. Inhibition was also observed under conditions where mannosyl-P -dolichol (Man-P-dol) stimulated the biosynthesis of GlcNAc-P-P-dolichol; t he apparent gi values for GlcNAc-P-P-dolichol and GlcNAc-GlcNAc-P-P-dolicho l were 2.2 and 11 mu M, respectively. Kinetic analysis of the types of inhi bition indicated competitive inhibition by GlcNAc-P-P-dolichol toward the s ubstrate UDP-GlcNAc and non-competitive inhibition toward dolichol phosphat e, Inhibition by GlcNAc-GlcNAc-P-P-dolichol was uncompetitive toward UDP-Gl cNAc and competitive toward dolichol phosphate, A model is presented for th e kinetic mechanism of the synthesis of GlcNAc-P-P-dolichol. GlcNAc-P-P-dol ichol also exerts a stimulatory effect on the biosynthesis of Man-P-dol, i, e, a reciprocal relationship to that previously observed between these two intermediates of the dolichol pathway. This network of inhibitory and stimu latory influences may be aspects of metabolic control of the pathway and th us of glycoprotein biosynthesis in general.