Structure and mechanism of galactose oxidase. The free radical site.
1994; Elsevier BV; Volume: 269; Issue: 40 Linguagem: Inglês
10.1016/s0021-9258(17)31504-1
ISSN1083-351X
AutoresAndrew J. Baron, Conrad Stevens, Carrie M. Wilmot, Kanjula Seneviratne, Veronica Blakeley, David M. Dooley, Simon E. V. Phillips, Peter F. Knowles, Michael J. McPherson,
Tópico(s)Porphyrin Metabolism and Disorders
ResumoCrystallographic 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 wildtype enzyme and mutational variants (W290H and C228G) have been characterized by x-ray crystallography, visible spectroscopy, and catalytic activity measurements.Afurther 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.Galactose oxidase (EC 1.1.3.9) is an extracellular enzyme produced by a Fusarium spp.' and is the simplest known copper-containing oxidase.Its structure and catalytic mechanism have been studied extensively using biochemical and spectroscopic methods (for review see Knowles and Ito, 1993).The x-ray structure to 1.7 h resolution has been reported for a crystal form at pH 4.5 (Ito et al., 1991).In addition the gaoA gene that encodes galactose oxidase has been characterized (McPherson et al., 1992b).Galactose oxidase catalyzes the stereospecific oxidation of D-isomers of a range of primary alcohol substrates, including D-galactose and polysaccharides with D-galactose at their nonreducing end, leading to the production of the corresponding
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