Synthetic substrate analogues for UDP-GlcNAc: Manα1-3R β1-2-N-acetylglucosaminyltransferase I. Substrate specificity and inhibitors for the enzyme

Abstract

UDP-GlcNAc:Manα1-3R β1-2-N-acetylglucosaminyltransferase I (GlcNAc-T I; EC 2.4.1.101) catalyses the conversion of [Manα1-6(Manα1-3)Manα1-6][Manα1-3]Manβ-O-R to [Manα1-6(Manα1-3)Manα1-6] [GlcNAcβ1-2Manα1-3]Manβ-O-R (R=1-4GlcNAcβ1-4GlcNAc-Asn-X) and thereby controls the conversion of oligomannose to complex and hybrid asparagine-linked glycans (N-glycans). GlcNAc-T I also catalyses the conversion of Manα1-6(Manα1-3)Manβ-O-octyl to Manα1-6(GlcNAcβ1-2Manα1-3)Manβ-O-octyl. We have therefore tested a series of synthetic analogues of Man″α1-6(Man′α1-3)Manβ-O-octyl as substrates and inhibitors for rat liver GlcNAc-T I. The 2″-deoxy and the 3″-, 4″- and 6″-O-methyl derivatives are all good substrates confirming previous observations that the hydroxyl groups of the Man″α1-6 residue do not play major roles in the binding of substrate to enzyme. In contrast, all four hydroxyl groups on the Man′α1-3 residue are essential since the corresponding deoxy derivatives either do not bind (2′- and 3′-deoxy) or bind very poorly (4′- and 6′-deoxy) to the enzyme. The 2′- and 3′-O-methyl derivatives also do not bind to the enzyme. However, the 4′-O-methyl derivative is a substrate (K _m =2.6mm) and the 6′-O-methyl compound is a competitive inhibitor (K _i=0.76mm). We have therefore synthesized various 4′- and 6′-O-alkyl derivatives, some with reactive groups attached to anO-pentyl spacer, and tested these compounds as reversible and irreversible inhibitors of GlcNAc-T I. The 6′-O-(5-iodoacetamido-pentyl) compound is a specific time dependent inhibitor of the enzyme. Four other 6′-O-alkyl compounds showed competitive inhibition while the remaining compounds showed little or no binding indicating that the electronic properties of the attachedO-pentyl groups influence binding.