Chemical modification of an α3-fucosyltransferase; definition of amino acid residues essential for enzyme activity

Abstract The biosynthesis of the carbohydrate antigen sialyl Lewis X (sLe x ) is dependent on the activity of an α 3-fucosyltransferase (EC 2.4.1.152, GDP-fucose:Gal β (1–4)GlcNAc-R α (1–3)fucosyltransferase). This enzyme catalyses the transfer of fucose from GDP- β -fucose to the 3-OH of N -acetylglucosamine present in lactosamine acceptors. In this report, we have investigated the amino acids essential for the activity of a recombinant α 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 IC 50 values of less than 200 μ M. Reagents selective for arginine and lysine had no effect on enzyme activity. The inclusion of GDP- β -fucose during preincubation with NEM reduces the rate of inactivation whereas inclusion of an acceptor saccharide for the enzyme, Gal β (1–4)GlcNAc, had no effect. No protective effect with either GDP- β -fucose or Gal β (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 site of the enzyme, FucT-VI possesses histidine residue(s) that are essential for enzyme activity.