The catalytic mechanism of the copper-containing enzyme galactose oxidase 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 axial copper ligand that is weakly co-ordinated to copper. In this study we have generated and studied the properties of a Y495F variant to test this proposal. X-ray crystallography reveals essentially no change from wild-type other than loss of the tyrosyl hydroxyl group. Visible spectroscopy indicates a significant change in the oxidised Y495F compared to wild-type with loss of a broad 810-nm peak, supporting the suggestion that this feature is due to inter-Iigand charge transfer via the copper. The presence of a peak at 420 nm indicates that the Y495F variant remains capable of radical formation, a fact supported by EPR measurements. Thus the significantly reduced catalytic efficiency (1100-fold lower k(cat)/K(m)) observed for this variant is not due to an inability to generate the Tyr272 radical. By studying azide-induced pH changes, it is clear that the reduced catalytic efficiency is due mainly to the inability of Y495F to accept protons. This provides definitive evidence for the key role of Tyr495 in the initial proton abstraction step of the galactose oxidase catalytic mechanism.
Bibliographical noteFunding Information:
Acknowledgements This work is supported by grants from the Biotechnology and Biology Research Council (BBSRC) and the Engineering and Physical Sciences Research Council and by BBSRC studentships to MPR and EV. SEVP is an International Research Scholar of the Howard Hughes Medical Institute. We thank D.M. Dooley and C.G. Saysell for helpful discussions and C.E. Ruggiero and M.S. Rogers for help with EPR simulations using the program kindly supplied by J.D. Lipscomb.
Copyright 2004 Elsevier Science B.V., Amsterdam. All rights reserved.
- Galactose oxidase
- Proton abstraction
- Tyrosine free radical