A sequence motif involved in the donor substrate binding by alpha1,6-fucosyltransferase: the role of the conserved arginine residues.
Alpha1,6-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 alpha1,6-fucosyltransferases in which the conserved residues in the region homologous to alpha1,2-fucosyltransferase had been replaced. These proteins were then characterized by kinetic analyses. The wild-type and mutant alpha1,6-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 K (m) value for both GDP-fucose and the acceptor oligosaccharide but did not markedly affect the apparent V (max). When these mutants were inhibited by GDP in a competitive manner with respect to the donor substrate, the K (i) 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 K (i) 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 beta-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 alpha1,2- and alpha1,6-fucosyltransferases is critical for binding of the donor substrate, GDP-fucose.
Takahashi
T
T
Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Ikeda
Y
Y
Tateishi
A
A
Yamaguchi
Y
Y
Ishikawa
M
M
Taniguchi
N
N
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D016367:Baculoviridae / Q000235:genetics; D001667:Binding, Competitive; D017124:Conserved Sequence; D005647:Fucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000494:pharmacology; D006154:Guanosine Diphosphate Fucose / Q000378:metabolism; D006157:Guanosine Monophosphate / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D018411:Spodoptera / Q000201:enzymology; D013329:Structure-Activity Relationship
D006153:Guanosine Diphosphate; D006154:Guanosine Diphosphate Fucose; D006157:Guanosine Monophosphate; D001120:Arginine; D005647:Fucosyltransferases; C020559:Glycoprotein 6-alpha-L-fucosyltransferase