Tyrosine 495 is a key residue in the active site of Galactose oxidase

Abstract

A 1.7A X-ray structure has been determined [2,3] and the structure and catalytic mechanism have been extensively studied (see [4,5] for reviews). The crystal structure shows that the enzyme contains a single cupric ion, coordinated in a pseudo square-planar geometry (Figure 1). The novel thioether bond [6] between Tyr272 and the adjacent Cys 228, together with the stacked aromatic ring of Trp290, have been suggested [2] to be involved in the stabilisation of the radical involved in catalysis [4]. It has been proposed [7] that the axial Tyr495 accepts a proton from bound substrate in the first step of the catalytic mechanism (Fig. 1). To investigate this proposal a mutational variant of GOase was prepared with Tyr495 replaced by Phe (Y495F). Aspergillus nidulans was used as a heterologous host for GOase expression. Mutagenesis was performed by a PCR approach with complete gene resequencing [8]. Preparation and purification of the Y495F mutant was performed as previously described for the wild-type enzyme [8]. Crystals of Y495F have been produced which diffract to 2.8A. The difference map compared to the wild-type structure [2,3] shows a slight shift in the F495 ring position, a compensating movement in the ring of H496 and a small shift of the Y272 ring. Atomic absorption spectrometry was carried out as previously described [8], showing 0.85 moles of copper per mole of enzyme, which is comparable to that seen in the wild-type enzyme. Steady-state kinetic studies on the ferricyanide-activated mutant enzyme gave values of Km=73.2i5.5 mM and k, =3.28&0.26 s.'. The Km is similar to that observed for wildtype enzyme and other mutants [8]. k J K I for this mutant is reduced 1100-fold compared to the wild-type enzyme. Visible absorption spectroscopy was performed on enzyme that was oxidised with hexachloroiridate and then desalted on a Biogel P6-DG column. Figure 2 shows a comparison of this spectrum with that obtained with native enzyme. The 445nm peak in GOase, has shifted to 410nm in the mutant, consistent with a loss of the Y495-C~'' RCH20H t 0 2 -+ RCHO t H202