A sequence motif involved in the donor substrate binding by 1,6-fucosyltransferase: the role of the conserved arginine residues
| Autor: | Akihiro Tateishi, Yoshitaka Ikeda, Yukihiro Yamaguchi, Tomoaki Takahashi, Naoyuki Taniguchi, Mutsuo Ishikawa |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Arginine
Stereochemistry Amino Acid Motifs Mutant Guanosine Monophosphate Spodoptera Binding Competitive Guanosine Diphosphate Biochemistry Fucose Kelch motif Structure-Activity Relationship chemistry.chemical_compound Guanosine Diphosphate Fucose Animals Humans Amino Acid Sequence Structural motif Conserved Sequence chemistry.chemical_classification Wild type Oligosaccharide Fucosyltransferases Kinetics chemistry Mutagenesis Site-Directed Sequence motif Baculoviridae |
| Zdroj: | Glycobiology. 10:503-510 |
| ISSN: | 1460-2423 0959-6658 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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