Control of glycoprotein synthesis: substrate specificity of rat liver UDP-GlcNAc:Man alpha 3R beta 2-N-acetylglucosaminyltransferase I using synthetic substrate analogues.
UDP-GlcNAc: Man alpha 3R beta 2-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). The Vmax 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 mumol 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 KM 20-fold. Modifications on the alpha 6-linked mannose or on the core structure affect mainly the KM and to a lesser degree the Vmax, 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 KM, whereas various other substitutions at the 3-position increase the KM slightly. Man alpha 6(Man alpha 3)4-O-methyl-Man beta 4GlcNAc was found to be a weak inhibitor of GlcNAc-T I.
Möller
G
G
Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Reck
F
F
Paulsen
H
H
Kaur
K J
KJ
Sarkar
M
M
Schachter
H
H
Brockhausen
I
I
D000818:Animals; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000096:biosynthesis*; D008099:Liver / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D008358:Mannose / Q000378:metabolism; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity
D006023:Glycoproteins; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I; D008358:Mannose