Acceptor-substrate recognition by N-acetylglucosaminyltransferase-V: Critical role of the 4″-hydroxyl group in β- d-Glc pNAc-(1 → 2)-α- d-Man p(1 → 6)-β- d-Glc p-OR

Abstract

The enzyme N-acetylglucosaminyltransferase-V (GlcNAcT-V) transfers GlcNAc from UDP-GlcNAc to the OH-6′ group of oligosaccharides terminating in the sequence β- d-Glc pNAc-(1 → 2)-α- d-Man p(1 → 6)-β- d-Glc p (or Man p)-OR ( 5, R  (CH 2) 7CH 3) to yield the sequence β- d-Glc pNAc-(1 → 2)-[β- d-Glc pNAc-(1 → 6)]-α- d-Man p-(1 → 6)-β- d-Glc p (or Man p)-OR. Biosynthetically, if β-(1 → 4)-galactosyltransferase acts first on 5, the product β- d-Gal p-(1 → 4)-β- d-Glc pNAc-(1 → 2)-α- d-Man p-(1 → 6)-β- d-Glc p (or Man p)-OR ( 7) is no longer a substrate for GlcNAcT-V even though it retains the active OH-6′ group. The reason for this loss in activity is examined in this paper. Six analogues of the acceptor trisaccharide 5, all with the reducing-end d- gluco configuration, were chemically synthesized. A key feature of the synthetic scheme is the use of 1,2-diaminoethane for the efficient removal of N-phthalimdo protecting groups. In these analogues OH-4 of the terminal sugar unit, the site of galactosylation by GalT in the normal GlcNAc-terminating trisaccharide 5, was systematically replaced by OMe, F, NH 2, NHAc, and H, as well as inverted to the galacto configuration. The interactions of the resulting trisaccharide analogues with GlcNAcT-V from hamster kidney were then evaluated kinetically. All six compounds were found to be essentially inactive either as acceptors or as inhibitors of GlcNAcT-V. The conclusion is reached that galactosylation of natural acceptors for GlcNAcT-V destroys acceptor activity, not by introduction of the steric bulk of an added sugar residue, but by destroying an important hydrogen-bonding interaction of terminal OH-4 of the GlcNAc residues with the enzyme. This OH-4 group is therefore designated as a key polar group for GlcNAcT-V.