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, a...

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Bibliographic Details
Published in:The Journal of biological chemistry 1994-10, Vol.269 (40), p.25095-25105
Main Authors: Baron, A.J. (The University of Leeds, Leeds, UK.), Stevens, C, Wilmot, C, Seneviratne, K.D, Blakeley, V, Dooley, D.M, Phillips, S.E.V, Knowles, P.F, McPherson, M.J
Format: Article
Language:English
Subjects:
ACIDE AMINE
ACTIVIDAD ENZIMATICA
ACTIVITE ENZYMATIQUE
AMINOACIDOS
ASPERGILLUS NIDULANS
Base Sequence
Binding Sites
COBRE
Crystallography, X-Ray
CUIVRE
Free Radicals
Fusarium
GALACTOSA
GALACTOSE
Galactose Oxidase - biosynthesis
Galactose Oxidase - chemistry
Galactose Oxidase - isolation & purification
HYPOMYCES
Kinetics
MINERALOGIA
MINERALOGIE
Molecular Sequence Data
MUTACION
MUTATION
OXIDORREDUCTASAS
OXYDOREDUCTASE
Polymerase Chain Reaction
RADICAL LIBRE
RADICALES LIBRES
TECHNIQUE ANALYTIQUE
TECNICAS ANALITICAS
TRANSFORMACION GENETICA
TRANSFORMATION GENETIQUE
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Description
Summary: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
ISSN:0021-9258
1083-351X
DOI:10.1016/s0021-9258(17)31504-1