Key involvement of all three GlcNAc hydroxyl groups in the recognition of β-D-Glc pNAc-(1→2)-α-D-Man p-(1→6)-β-D-Glc p-OR by N-acetylglucosaminyltransferase-V

Abstract

N-Acetylglucosammyltransferase-V (GlcNAc T-V) transfers a β-linked GlcNAc residue from UDP-GlcNAc to OH-6′ (of the αMan residue) in oligosaccharides terminating in the sequence β- d-Gl cp NAc-(1→2)-α- d-Man p-(1→)-β- d-Glc p (or Man ( 3, R = (CH 2) 7CH 3). It was previously found that OH-4″ (of the GlcNAc residue) in 3 was a critical element for substrate recognition by this enzyme. We show here that OH-3″ and OH-6″ are also key recognition elements. Four analogs of trisaccharide 3 where OH-3″ and OH-6″ were replaced, independently, by NH 2 and NHAc groups, were prepared by multi-step chemical synthesis and kinetically evaluated as substrates for GlcNAc T-V from hamster kidney. These substitutions were selected since they replaced the OH groups with groups probing both hydrogen bonding and steric bulk. The 3″-modified compounds were found to be very poor substrates with K m values more than 50-fold elevated over that for 3 (26 μM) while the 6″-modified compounds were completely inactive. An intact 3,4,6 triol system in the terminal GlcNAc residue therefore appears to be the key polar group system that is recognized by this enzyme.