title: Site-directed mutation of the active site of influenza neuraminidase and implications for the catalytic mechanism.
abstract: Different isolates of influenza virus show a high degree of amino acid sequence variation in their surface glycoproteins. Conserved residues located in the substrate-binding pocket of the influenza virus neuraminidase are therefore likely to be involved in substrate binding or enzyme catalysis. In order to study the structure and function of the active site of this protein, a full-length cDNA clone of the neuraminidase gene from influenza A/Tokyo/3/67 was subcloned into aN M13 vector and amino acid substitutions were made in selected residues by using the oligonucleotide mismatch technique. The mutant neuraminidase genes were expressed from a recombinant SV40 vector, and the proteins were analyzed for synthesis, transport to the cell surface, and proper three-dimensional folding by internal and surface immunofluorescence. The mutant neuraminidase proteins were then assayed to determine the effect of the amino acid substitution on enzyme activity. Twelve of the 14 mutant proteins were correctly folded and were transported to the cell surface in a manner identical with that of the wild type. Two of these have full enzyme activity, but seven mutants, despite correct three-dimensional structure, have completely lost neuraminidase activity. Two mutants were active at low pH. The properties of the mutant enzymes suggest a possible mechanism of neuraminidase action.
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, 35294.
authors.forename: M R
authors.initials: MR
authors.lastname: Lentz
authors.forename: R G
authors.initials: RG
authors.lastname: Webster
authors.forename: G M
authors.initials: GM
authors.lastname: Air
mesh_terms: D001665:Binding Sites; D003001:Cloning, Molecular; D005455:Fluorescent Antibody Technique; D009980:Influenza A virus / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D009439:Neuraminidase / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation
chemical_list: D009439:Neuraminidase