CONTROL OF GLYCOPROTEIN-SYNTHESIS - SUBSTRATE-SPECIFICITY OF RAT-LIVER UDP-GLCNAC-MAN ALPHA-3R BETA-2-N-ACETYLGLUCOSAMINYL-TRANSFERASE-I USING SYNTHETIC SUBSTRATE-ANALOGS

UDP-GlcNAc: Man-alpha-3R beta-2-N-acetylglucosaminyltransferase I (GlcNAc-TI; EC 2.4.1.101) is the key enzyme in the synthesis of complex and hybrid N-glycans. Rat liver GlcNAc-T I has been purified more than 25,000-fold (M(r) 42,000). The V(max) for the pure enzyme with [Man-alpha-6(Man-alpha-3)Man-alpha-6](Man-alpha-3)Man-beta-4GlcNAc-beta-4GlcNAc-beta-Asn as substrate was 4.6-mu-mol min-1 mg-1. Structural analysis of the enzyme product by proton nuclear magnetic resonance spectroscopy proved that the enzyme adds an N-acetylglucosamine (GlcNAc) residue in beta-1-2 linkage to the Man-alpha-3Man-beta-terminus of the substrate. Several derivatives of Man-alpha-6(Man-alpha-3)Man-beta-R, a substrate for the enzyme, were synthesized and tested as substrates and inhibitors. An unsubstituted equatorial 4-hydroxyl and an axial 2-hydroxyl on the beta-linked mannose of Man-alpha-6(Man-alpha-3)Man-beta-R are essential for GlcNAc-T I activity. Elimination of the 4-hydroxyl of the alpha-3-linked mannose (Man) of the substrate increases the K(M) 20-fold. Modifications on the alpha-6-linked mannose or on the core structure affect mainly the K(M) and to a lesser degree the V(max), e.g., substitutions of the Man-alpha-6 residue at the 2-position by GlcNAc or at the 3- and 6-positions by mannose lower the K(M), whereas various other substitutions at the 3-position increase the K(M) slightly. Man-alpha-6(Man-alpha-3)4-O-methyl-Man-beta-4GlcNAc was found to be a weak inhibitor of GlcNAc-T I.