Crystal Structure of a Free Radical Enzyme, Galactose Oxidase

• Published in: Journal of molecular biology Vol. 238; no. 5; pp. 794 - 814
• Main Authors: Ito, Nobutoshi, Phillips, Simon E.V., Yadav, Kapil D.S., Knowles, Peter F.
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 20.05.1994; Elsevier
• Subjects: alcohol oxidoreductases; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Binding Sites; Biological and medical sciences; Computer Graphics; copper; Copper - chemistry; copper-containing enzyme; Crystalline structure; Crystallization; Crystallography, X-Ray; Enzymes and enzyme inhibitors; Free Radicals; Fundamental and applied biological sciences. Psychology; Fusarium; Fusarium - chemistry; galactose oxidase; Galactose Oxidase - chemistry; Hypomyces rosellus; Models, Biological; Models, Molecular; Molecular biophysics; molecular conformation; Molecular Sequence Data; Molecular Structure; Oxidoreductases; pdb/1glw; Protein Structure, Tertiary; Sequence Alignment; structure; Structure in molecular biology; Tyrosine - chemistry; tyrosine free radical; tyrosine-cysteine bridge; X-ray crystal structure; X-ray diffraction; tyrosine free radical; X-ray crystal structure; copper-containing enzyme; galactose oxidase; tyrosine-cysteine bridge; Tyrosine; Ligand binding; Enzyme; Active site; Refinement; Secondary structure; Free radical; Substrate; Galactose oxidase; Copper Ions; Models; Oxidoreductases; Structural analysis; Crystalline structure
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.1994.1335

The crystal structure of the copper-containing enzyme, galactose oxidase, has been solved by multiple isomorphous replacement and refined to a resolution of 1·7 Å. The X-ray structure reveals a unique polypeptide fold. The protein can be divided into three domains, all of which consist almost entirely of β-strands. The structure of the second domain is particularly striking, 28 β-strands arranged in a pseudo 7-fold symmetry. The copper site is on the surface of the protein and extremely rich in aromatic side-chains. The copper ion has two histidine, two tyrosines, and one external ligand in distorted square pyramidal coordination. The presence of pyrroloquinoline quinone as a covalently bound cofactor in GOase has been excluded. Instead, an unexpected covalent linkage between Tyr272 and Cys228 has been observed, whose functional role may relate to the presence of a tyrosine free radical at Tyr272. The tyrosine free radical could be stabilized by delocalization to Cys228 and stacking interactions with Trp290. A structural model for substrate binding is proposed that offers an explanation for the substrate specificity of the enzyme and many of the spectroscopic and enzymological data. Although the model lacks direct confirmation at present, it should provide a stimulus for further spectroscopic and crystallographic studies.