Chemical modification of an alpha 3-fucosyltransferase; Definition of amino acid residues essential for enzyme activity

The biosynthesis of the carbohydrate antigen sialyl Lewis X (sLe(x)) is dependent on the activity of an alpha 3-fucosyltransferase (EC 2.4.1.152, GDP-fucose:Gal beta(1-4)GlcNAc-R alpha(1-3)fucosyltransferase). This enzyme catalyses the transfer of fucose from GDP-beta-fucose to the 3-OH of N-acetylglucosamine present in lactosamine accepters. In this report, we have investigated the amino acids essential for the activity of a recombinant alpha 3-fucosyltransferase (FucT-VI) through chemical modification of the enzyme with group-selective reagents. FucT-VI activity was found to be particularly sensitive to the histidine-selective reagent diethylpyrocarbonate and the cysteine reagent N-ethylmaleimide, with IC50 values of less than 200 mu M. Reagents selective for arginine and lysine had no effect on enzyme activity. The inclusion of GDP-beta-fucose during preincubation with NEM reduces the rate of inactivation whereas inclusion of an acceptor saccharide for the enzyme, Gal beta(1-4)GlcNAc, had no effect. No protective effect with either GDP-beta-fucose or Gal beta(1-4)GlcNAc was observed on treatment of the enzyme with diethylpyrocarbonate. These data suggest that in addition to an NEM-reactive cysteine in, or adjacent to. the substrate-binding sire of the enzyme, FucT-VI possesses histidine residue(s) that are essential for enzyme activity.