STRUCTURE AND MECHANISM OF GALACTOSE-OXIDASE - THE FREE-RADICAL SITE

Crystallographic and spectroscopic studies on galactose oxidase have s hown that the active site involves a free radical on tyrosine 272, one of the ligands coordinated to the Cu2+ cofactor. A novel thioether bo nd between tyrosine 272 and cysteine 228, and a stacking tryptophan 29 0, over this bond, are features of the crystal structure. The present study describes the development of a high level heterologous expressio n system for galactose oxidase and the construction of mutational vari ants at these key active site residues. The expressed wildtype enzyme and mutational variants (W290H and C228G) have been characterized by x -ray crystallography, visible spectroscopy, and catalytic activity mea surements. A further variant protein, Y272F, could not be purified. Th e data establish that the thioether bond and stacking tryptophan are e ssential for activity and further support a role for tryptophan 290 as a component of the free radical site.