Control of glycoprotein synthesis: substrate specificity of rat liver UDP-GlcNAc:Manα3R β2-N-acetylglucosaminyl-transferase I using synthetic substrate analogues

Abstract

UDP-GlcNAc: Manα3R β2-N-acetylglucosaminyltransferase I (GlcNAc-T I; 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). TheV _max for the pure enzyme with [Manα6(Manα3)Manα6](Manα3)Manβ4GlcNAcβ4GlcNAcβ-Asn as substrate was 4.6 µmol min⁻¹ mg⁻¹. Structural analysis of the enzyme product by proton nuclear magnetic resonance spectroscopy proved that the enzyme adds anN-acetylglucosamine (GlcNAc) residue in β1–2 linkage to the Manα3Manβ-terminus of the substrate. Several derivatives of Manα6(Manα3)Manβ-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 β-linked mannose of Manα6(Manα3)Manβ-R are essential for GlcNAc-T I activity. Elimination of the 4-hydroxyl of the α3-linked mannose (Man) of the substrate increases theK _M 20-fold. Modifications on the α6-linked mannose or on the core structure affect mainly theK _M and to a lesser degree theV _max, e.g., substitutions of the Manα6 residue at the 2-position by GlcNAc or at the 3- and 6-positions by mannose lower theK _M, whereas various other substitutions at the 3-position increase theK _M slightly. Manα6(Manα3)4-O-methyl-Manβ4GlcNAc was found to be a weak inhibitor of GlcNAc-T I.