SUBSTRATE COMPLEXES OF THE FREE RADICAL ENZYME GALACTOSE OXIDASE
Elinor Vinecombe & Simon E. V. Phillips.
School of Biochemistry and Molecular Biology, University
of Leeds, Leeds, LS2 9JT, United Kingdom.
vinecombe@bmb.leeds.ac.uk
Keywords: Galactose oxidase, Copper
Galactose oxidase is a 68kDa monomeric enzyme produced by Fusaria spp. that catalyses the oxidation of primary alcohols to their corresponding aldehydes. It contains a single copper(II) ion with a protein free radical cofactor, situated on copper ligand Tyr272, acting as a second redox centre. Although stereo-specific, galactose oxidase has a wide substrate specificity, ranging from small sugars to polysaccharides. The x-ray crystal structure has been determined to 1.7 A resolution (1).
The enzyme crystallises in the space group C2, such that the active sites of symmetry-related molecules occlude one another. For this reason substrates, such as galactose, are unable to bind in the active site once the enzyme has been crystallised. However, in the absence of crystal structures of enzyme- substrate complexes, a model for substrate binding has been proposed (2), with D-galactose binding with the 6-hydroxyl oxygen in an equatorial position to the copper, displacing a water molecule which is found in the native structure.
We have produced a new crystal form by crystallizing wild-type enzyme, treated with the chelating agent sodium diethyldithiocarbamate. This copper free form crystallizes in the orthorhombic space group P212121 with cell dimensions a=59.8, b=89.8, c=135.7 A . A room temperature data set to 2.7 A has been collected and the structure solved by molecular replacement. In form there appears to be room for a substrate molecule to bind in the active site. The model is this currently being refined and these results, along with those from crystal soaking studies will be presented.
Since it will probably be possible to soak copper back into the crystals, this new crystal form should allow us to study the binding of substrates and inhibitors in the crystal by galactose oxideas and further the understanding of its catalytic mechanism.