STRUCTURE AND MECHANISM OF GALACTOSE-OXIDASE - CATALYTIC ROLE OF TYROSINE-495

The catalytic mechanism of the copper-containing enzyme galactose oxid ase involves a protein radical on Tyr272, one of the equatorial copper ligands. The first step in this mechanism has been proposed to be the abstraction of a proton from the alcohol substrate by Tyr495, the axi al copper ligand that is weakly co-ordinated to copper. In this study we have generated and studied the properties of a Y495F variant to tes t this proposal. X-ray crystallography reveals essentially no change f rom wild-type other than toss of the tyrosyl hydroxyl group. Visible s pectroscopy indicates a significant change in the oxidised Y495F compa red to wildtype with loss of a broad 810-nm peak, supporting the sugge stion that this feature is due to inter-ligand charge transfer via the copper. The presence of a peak at 420 nm indicates that the Y495F var iant remains capable of radical formation, a fact supported by EPR mea surements. Thus the significantly reduced catalytic efficiency (1100-f old lower k(cat)/K-m) observed for this variant is not due to an inabi lity to generate the Tyr272 radical. By studying azide-induced pH chan ges, it is clear that the reduced catalytic efficiency is due mainly t o the inability of Y495F to accept protons. This provides definitive e vidence for the key role of Tyr495 in the initial proton abstraction s tep of the galactose oxidase catalytic mechanism.