title: Site-directed mutagenesis of the catalytic residues Asp-52 and Glu-35 of chicken egg white lysozyme.
abstract: The roles of the catalytic active-site residues aspartic acid-52 and glutamic acid-35 of chicken lysozyme (EC 3.2.1.17) have been investigated by separate in vitro mutagenesis of each residue to its corresponding amide (denoted as D52N and E35Q, respectively). The mutant enzyme D52N exhibits approximately 5% of the wild-type lytic activity against Micrococcus luteus cell walls, while there is no measurable activity associated with E35Q (0.1% +/- 0.1%). The measured dissociation constants for the chitotriose-enzyme complexes were 4.1 microM (D52N) and 13.4 microM (E35Q) vs. 8.6 microM for wild type, indicating that the alterations in catalytic properties may be due in part to binding effects as well as to direct catalytic participation of these residues. The mutant lysozymes have been expressed in and secreted from yeast and obtained at a level of approximately 5 mg per liter of culture by high-salt elution from the cell walls.
authors.affiliation: Department of Biochemistry, University of California, Berkeley.
authors.forename: B A
authors.initials: BA
authors.lastname: Malcolm
authors.forename: S
authors.initials: S
authors.lastname: Rosenberg
authors.forename: M J
authors.initials: MJ
authors.lastname: Corey
authors.forename: J S
authors.initials: JS
authors.lastname: Allen
authors.forename: A
authors.initials: A
authors.lastname: de Baetselier
authors.forename: J F
authors.initials: JF
authors.lastname: Kirsch
mesh_terms: D000818:Animals; D001224:Aspartic Acid; D001483:Base Sequence; D001665:Binding Sites; D002645:Chickens; D003001:Cloning, Molecular; D004529:Egg White; D005971:Glutamates; D018698:Glutamic Acid; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D005971:Glutamates; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D009113:Muramidase