Glycobiology, 2000, Vol. 10, No. 5 503-510
© 2000 Oxford University Press
A sequence motif involved in the donor substrate binding by 
1,6-fucosyltransferase: the role of the conserved arginine residues
2Department of Biochemistry, Osaka University Medical School, 2–2 Yamadaoka, Suita, Osaka 565–0871, Japan and 3Department of Obstetrics and Gynecology, Asahikawa Medical College, Nishikagura 4–5–3–11, Asahikawa, Hokkaido 078–8510, Japan
1,6-Fucosyltransferase catalyzes the transfer of fucose to the innermost GlcNAc residue of an N-linked oligosaccharide. In order to identify the amino acid residue(s) which are associated with the enzyme activity and to investigate their function, we prepared a series of mutant human 1,6-fucosyltransferases in which the conserved residues in the region homologous to 1,2-fucosyltransferase had been replaced. These proteins were then characterized by kinetic analyses. The wild-type and mutant 1,6-fucosyltransferases were expressed using a baculovirus-insect cell system. The activity assay showed that replacement of Arg-365 by Ala or Lys led to a complete loss of activity while substitution of Ala or Lys for the neighboring Arg-366 decreased the activity to about 3% that of the wild type. Kinetic analyses revealed that the replacements of Arg-366 lead to an increase in the apparent Km value for both GDP-fucose and the acceptor oligosaccharide but did not markedly affect the apparent Vmax. When these mutants were inhibited by GDP in a competitive manner with respect to the donor substrate, the Ki values were found to be 50–100 times higher than the value in the wild type. On the other hand, in the inhibition by GMP, the Ki values for the mutants were very similar to that of the wild type. These findings suggest that Arg-366 contributes to the binding of GDP-fucose via an interaction with the ß-phosphoryl group of the GDP moiety of the donor, and that Arg-365 may also play an essential role in substrate binding. The results suggest that the motif common to 1,2- and 1,6-fucosyltransferases is critical for binding of the donor substrate, GDP-fucose.
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