Chemical modifications of alpha1,6-fucosyltransferase define amino acid residues of catalytic importance.
alpha1,6-Fucosyltransferase (alpha6FucT) of human platelets was subjected to the action of phenylglyoxal (PLG), pyridoxal-5'-phosphate/NaBH(4) (PLP), and diethyl pyrocarbonate (DEPC) the reagents that selectively modify the structure of amino acids arginine, lysine and histidine, respectively, as well as to N-ethylmaleimide (NEM), mersalyl, p-chloromercuribenzoate (pCMB), iodoacetate, iodoacetamide, and methyl iodide that react with sulfhydryl group of cysteine. In addition, we treated the enzyme with beta-mercaptoethanol, a reagent that disrupts disulfide bonds. All reagents except NEM significantly inactivated alpha6FucT. Protection against the action of PLG, PLP and sulfhydryl modifying reagents was offered by GDP-fucose, GDP, and the acceptor substrate, a transferrin-derived biantennary glycopeptide with terminal GlcNAc residues. Neither donor nor acceptor substrate offered, however, any protection against inactivation by DEPC or beta-mercaptoethanol. We conclude that arginine, cysteine and probably lysine residues are present in, or closely by, the donor and acceptor substrate binding domains of the enzyme, whereas histidine may be a part of its catalytic domain. However, the primary structure of alpha6FucT does not show cysteine residues in proximity to the postulated GDP-fucose-binding site and acceptor substrate binding site of the enzyme that contains two neighboring arginine residues and one lysine residue (Glycobiol. 10 (2000) 503). To rationalize our results we postulate that platelet alpha6FucT is folded through disulfide bonds that bring together donor/acceptor-binding- and cysteine- and lysine-rich, presumably acceptor substrate binding sites, thus creating a catalytic center of the enzyme.
Kamińska
J
J
Department of Biochemistry, Institute of Hematology and Blood Transfusion, Chocimska 5, 00-957 Warsaw, Poland.
Wiśniewska
A
A
Kościelak
J
J
D000596:Amino Acids / Q000737:chemistry; D001792:Blood Platelets / Q000201:enzymology; D020134:Catalytic Domain; D005647:Fucosyltransferases / Q000037:antagonists & inhibitors / Q000097:blood / Q000737:chemistry*; D006150:Guanine Nucleotides / Q000494:pharmacology; D006151:Guanosine / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007700:Kinetics; D017510:Protein Folding
D000596:Amino Acids; D006150:Guanine Nucleotides; D007202:Indicators and Reagents; D006151:Guanosine; D005647:Fucosyltransferases; C020559:Glycoprotein 6-alpha-L-fucosyltransferase