RRC ID 39951
著者 Wang Y, Yamaguchi K, Shimada Y, Zhao X, Miyagi T.
タイトル Site-directed mutagenesis of human membrane-associated ganglioside sialidase: identification of amino-acid residues contributing to substrate specificity.
ジャーナル Eur J Biochem
Abstract Unlike microbial sialidases, mammalian sialidases possess strict substrate specificity, for example the human membrane-associated sialidase, which hydrolyzes only gangliosides. To cast light on the molecular basis of this narrow substrate preference, predicted active site amino-acid residues of the human membrane sialidase were altered by site-directed mutagenesis. When compared with the active site amino-acid residues proposed for Salmonella typhimurium sialidase, only five out of 13 residues were found to be different to the human enzyme, these being located upstream of the putative transmembrane region. Alteration of seven residues, including these five, was followed by transient expression of the mutant enzymes in COS-1 cells and characterization of their kinetic properties using various substrates. Substitution of glutamic acid (at position 51) by aspartic acid and of arginine (at position 114) by glutamine or alanine resulted in retention of good catalytic efficiency toward ganglioside substrates, whereas other substitutions caused a marked reduction. The mutant enzyme E51D exhibited an increase in hydrolytic activity towards GM2 as well as sialyllactose (which are poor substrates for the wild-type) with change to a lower Km and a higher Vmax. R114Q demonstrated a substrate specificity shift in the same direction as E51D, whereas R114A enhanced the preference for gangliosides GD3 and GD1a that are effectively hydrolyzed by the wild-type. The inhibition experiments using 2-deoxy-2,3-didehydro-N-acetylneuraminic acid were consistent with the results in the alteration of substrate specificity. The findings suggest that putative active-site residues of the human membrane sialidase contribute to its substrate specificity.
巻・号 268(8)
ページ 2201-8
公開日 2001-4-1
DOI 10.1046/j.1432-1327.2001.02069.x
PII ejb2069
PMID 11298736
MeSH Amino Acid Sequence Amino Acids / chemistry Animals Arginine / chemistry Aspartic Acid / chemistry Binding Sites Blotting, Western COS Cells Cell Membrane / metabolism* Gangliosides / metabolism* Glutamic Acid / chemistry Glutamine / chemistry Humans Hydrogen-Ion Concentration Hydrolysis Kinetics Molecular Sequence Data Mutagenesis, Site-Directed Neuraminidase / chemistry* Neuraminidase / genetics Neuraminidase / metabolism Rats Salmonella typhimurium / enzymology Sequence Homology, Amino Acid Substrate Specificity Temperature Transfection
引用数 30
WOS 分野 BIOCHEMISTRY & MOLECULAR BIOLOGY
リソース情報
ヒト・動物細胞 COS-1(RCB0143)