Structure and mechanism of galactose oxidase. The free radical site

J Biol Chem. 1994 Oct 7;269(40):25095-105.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Binding Sites
  • Crystallography, X-Ray
  • Free Radicals
  • Galactose Oxidase / biosynthesis
  • Galactose Oxidase / chemistry*
  • Galactose Oxidase / isolation & purification
  • Kinetics
  • Molecular Sequence Data
  • Mutation
  • Polymerase Chain Reaction


  • Free Radicals
  • Galactose Oxidase