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 acceptors. 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 microM. 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 site of the enzyme, FucT-VI possesses histidine residue(s) that are essential for enzyme activity.
Britten
C J
CJ
Glycobiology Research Group, Glaxo Wellcome Research and Development Limited, Stevenage, Herts, UK. cjb14610@ggr.co.uk
Bird
M I
MI
D000595:Amino Acid Sequence; D003545:Cysteine / Q000032:analysis*; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005647:Fucosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D006154:Guanosine Diphosphate Fucose / Q000494:pharmacology; D006639:Histidine / Q000032:analysis*; D006801:Humans; D008969:Molecular Sequence Data; D016415:Sequence Alignment
D006154:Guanosine Diphosphate Fucose; D006639:Histidine; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase; D003545:Cysteine; D004047:Diethyl Pyrocarbonate; D005033:Ethylmaleimide