Abstract
The assembly of the core oligosaccharide region of asparagine-linked glycoproteins proceeds by means of the dolichol pathway. The first step of this pathway, the reaction of dolichol phosphate with UDP-GlcNAc to form N-acetylglucosaminylpyrophosphoryldolichol (GlcNAc-P-P-dolichol), is under investigation as a possible site of metabolic regulation. This report describes feedback inhibition of this reaction by the second intermediate of the pathway, N-acetylglucosaminyl-N-acetylglucosaminylpyrophosphoryldolichol (GlcNAc-GlcNAc-P-P-dolichol), and product inhibition by GlcNAc-P-P-dolichol itself. These influences were revealed when the reactions were carried out in the presence of showdomycin, a nucleoside antibiotic, present at concentrations that block the de novo formation of GlcNAc-GlcNAc-P-P-dolichol but not that of GlcNAc-P-P-dolichol. The apparent K(i) values for GlcNAc-P-P-dolichol and GlcNAc-GlcNAc-P-P-dolichol under basal conditions were 4.4 and 2.8 microM, respectively. Inhibition was also observed under conditions where mannosyl-P-dolichol (Man-P-dol) stimulated the biosynthesis of GlcNAc-P-P-dolichol; the apparent K(i) values for GlcNAc-P-P-dolichol and GlcNAc-GlcNAc-P-P-dolichol were 2.2 and 11 microM, respectively. Kinetic analysis of the types of inhibition indicated competitive inhibition by GlcNAc-P-P-dolichol toward the substrate UDP-GlcNAc and non-competitive inhibition toward dolichol phosphate. Inhibition by GlcNAc-GlcNAc-P-P-dolichol was uncompetitive toward UDP-GlcNAc and competitive toward dolichol phosphate. A model is presented for the kinetic mechanism of the synthesis of GlcNAc-P-P-dolichol. GlcNAc-P-P-dolichol 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 stimulatory influences may be aspects of metabolic control of the pathway and thus of glycoprotein biosynthesis in general.
Highlights
It has been well established that the dolichol pathway is the means whereby the core region of asparagine-linked glycoproteins is assembled
Our understanding of the mechanisms that regulate this complex series of reactions, is still limited. To this end we have directed our attention to the initial reaction of the pathway, the reaction between dolichol phosphate and UDP-GlcNAc producing GlcNAc-P-P-dolichol,1 catalyzed by the enzyme, UDP-GlcNAc:dolichyl-phosphate N-acetylglucosamine 1-phosphate transferase (GPT-1)
We have examined the effect of these inhibitory influences on the biosynthesis of GlcNAc-P-P-dolichol at the basal level and under stimulatory conditions in the presence of Man-P-dol
Summary
Microsomes were prepared from the retinas of 15–16-day-old embryonic chicks as described previously [10]. Effect of GlcNAc-P-P-dolichol on Man-P-Dol Formation—Embryonic chick retina microsomes were incubated at 37 °C for 15 min in a medium containing GDP-[3H]mannose (1.9 M, 200 dpm/pmol), dolichol phosphate (16 M), MnCl2 (20 mM), Triton X-100 (0.2%), Tes buffer (0.2 M, pH 7.45), in the presence or absence of GlcNAc-P-P-dolichol (as indicated), and enzyme in a total volume of 0.15 ml, as described previously [24]. Incubations were carried out at 37 °C for 10 min essentially as described by Schutzbach et al [25] in a medium containing 0.5% Nonidet P-40 (w/v), 25 mM Tris-HCl, pH 7.5, 5 mM MnCl2, 2.5 mM MgCl2, 0.25 mM EDTA, 5 mM dithiothreitol, 32 M dolichol phosphate, GDP[3H]Man (18 M, 12–22 dpm/pmol) in the presence or absence of GlcNAc-P-P-dolichol (as indicated) and enzyme in a total volume of 0.15 ml. Apparent Ka and Vmax values were calculated from Lineweaver-Burk double-reciprocal plots of the data after analysis by computer using the Kcat program (BioMetalics, Princeton, NJ)
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