Structure and mechanism of galactose oxidase. The free radical site
1994
Baron, A.J. | Stevens, C. | Wilmot, C. | Seneviratne, K.D. | Blakeley, V. | Dooley, D.M. | Phillips, S.E.V. | Knowles, P.F. | McPherson, M.J.
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.
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