A trisaccharide acceptor analog for N-acetylglucosaminyltransferase V which binds to the enzyme but sterically precludes the transfer reaction.

Development of inhibitors specific for the glycosyltransferases involved in the biosynthesis of asparagine-linked sugar chains has been undertaken in the hopes that these compounds may serve as tools to elucidate the roles of complex carbohydrates in biological recognition events. We report here the first example of a glycosyltransferase acceptor analog in which strategic replacement of a nonreacting hydroxyl group with a larger substituent produces a molecule which is recognized by the enzyme but does not react because of a steric block to the glycosyl transfer reaction. N-Acetylglucosaminyltransferase V catalyzes the transfer of GlcNAc from the sugar nucleotide donor UDP-GlcNAc to the 6-OH group of mannose in the synthetic trisaccharide acceptor beta GlcNAc(1-->2)alpha Man(1-->6)beta Glc-O(CH2)7CH3 (Km = 23 +/- 2 microM; Vmax = 116 +/- 3 pmol/h) to form the tetrasaccharide beta GlcNAc(1-->2)(beta GlcNAc(1-->6))alpha Man(1-->6)beta Glc-O(CH2)7CH3. The acceptor analog produced by replacement of the adjacent nonreacting 4-OH group of the mannose residue with an O-methyl group was not a substrate for the enzyme but was found to be a good competitive inhibitor of GlcNAc transferase V with Ki = 14 +/- 2 microM. To test the theory that it was the presence of the large methyl group which prevented the glycosyl transfer reaction the 4'-deoxygenated analog was synthesized. It was found to be a good substrate with Km = 74 +/- 6 microM and an almost 5-fold higher kcat (Vmax = 535 +/- 13 pmol/h). NMR data show no evidence of important conformational differences between the trisaccharide analogs, and kinetic experiments detected no differences for the binding of UDP-GlcNAc in their presence. The conclusion was therefore reached that the large methyl group introduced on O-4' sterically prevented the formation of product even though both potential substrates were bound by the enzyme. This "steric exclusion" strategy offers potential for the design of inhibitors for that class of glycosyltransferases in which the reactive hydroxyl group is also an essential recognition element.