title: Structure and mechanism of galactose oxidase. The free radical site.
abstract: Crystallographic and spectroscopic studies on galactose oxidase have shown that the active site involves a free radical on tyrosine 272, one of the ligands coordinated to the Cu2+ cofactor. A novel thioether bond between tyrosine 272 and cysteine 228, and a stacking tryptophan 290, over this bond, are features of the crystal structure. The present study describes the development of a high level heterologous expression system for galactose oxidase and the construction of mutational variants at these key active site residues. The expressed wild-type enzyme and mutational variants (W290H and C228G) have been characterized by x-ray crystallography, visible spectroscopy, and catalytic activity measurements. A further variant protein, Y272F, could not be purified. The data establish that the thioether bond and stacking tryptophan are essential for activity and further support a role for tryptophan 290 as a component of the free radical site.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Leeds, United Kingdom.
authors.forename: A J
authors.initials: AJ
authors.lastname: Baron
authors.forename: C
authors.initials: C
authors.lastname: Stevens
authors.forename: C
authors.initials: C
authors.lastname: Wilmot
authors.forename: K D
authors.initials: KD
authors.lastname: Seneviratne
authors.forename: V
authors.initials: V
authors.lastname: Blakeley
authors.forename: D M
authors.initials: DM
authors.lastname: Dooley
authors.forename: S E
authors.initials: SE
authors.lastname: Phillips
authors.forename: P F
authors.initials: PF
authors.lastname: Knowles
authors.forename: M J
authors.initials: MJ
authors.lastname: McPherson
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D005609:Free Radicals; D005692:Galactose Oxidase / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction
chemical_list: D005609:Free Radicals; D005692:Galactose Oxidase