96 On the role of manganese cation in the mechanism of α-1,3-fucosyltransferase

Glycosyl transferases are involved in the formation of many biologically important oligosaccharides. The mechanism of glycosyl transfer is not fully understood, but for some transferases the rate of in vitro glycosyl transfer can be enhanced by the addition of diva!-,nt cations [l]. The role of such cations, typically MnZ+, is unclear but it has been proposed that they may co-ordinate the pyrophosphate group of the sugar nucleotide donor substrate. Studies by Imperiali and co-workers [2] however, have shown that for oligosaccharyl transferase this may not be the case, and in fact it is believed that the divalent cation is in close proximity to the acceptor substrate. Whilst conducting studies with a series of systematically modified (sulfated) N-acetyllactosamine derivatives [3] and a1,3-fucosyltransferase (milk), we made an unexpected observation. Sulfation of the acceptor substrate at the site to which fucose is transferred by a-1,3-fucosyltransferase, did not render the compound inactive. In fact, kinetic studies showed the compound to be a good substrate for the enzyme. This led us to conclude that the enzyme may be capable of transferring fucose onto the sulfate moiety, thereby generating a labile glycosyl sulfate diester product. With a view to establishing support for this type of compound, we chose to prepare the corresponding N-acetyllactosamine phosphate derivative. This was based on the assumption that any resulting phosphate diester product would be chemically stable. Preliminary electrospray mass spectrometry data suggests that a phosphorylated, fucosylated derivative is obtained as a product of the enzyme reaction. Kinetic data for sulfated and phosphorylated Nacetyllactosamine compounds with recombinant a1,3fucosyltransferase VI are shown in Figure 1.