Structure and mechanism of galactose oxidase. The free radical site.
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.
Baron
A J
AJ
Department of Biochemistry and Molecular Biology, University of Leeds, United Kingdom.
Stevens
C
C
Wilmot
C
C
Seneviratne
K D
KD
Blakeley
V
V
Dooley
D M
DM
Phillips
S E
SE
Knowles
P F
PF
McPherson
M J
MJ
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
D005609:Free Radicals; D005692:Galactose Oxidase