Structural Studies on the Copper Sites in Benzylamine Oxidase from Pig Plasma

Abstract

Benzylamine oxidase (EC 1.4.3.4), homogeneous according to ultracentrifugation and disc gel electrophoresis in the presence and in the absence of sodium dodecyl sulphate, has a molecular weight of approx. 190000 as determined by ultracentrifugation to equilibrium (Yphantis, 1964). In 6~-guanidinium chloride, pH6.5, containing 0.1 Mp-mercaptoethanol and in 1 % sodium dodecyl sulphate, pH7.0, the enzyme is dissociated into subunits of molecular weight approx. 95000. These results were obtained with both equilibrium ultracentrifugation and disc gel electrophoresis in sodium dodecyl sulphate. The copper content/mol of enzyme is 2.1 +0.2 g-atoms determined chemically (Van de Bogart & Beinert, 1967) and approx. 2.3g-atoms determined by integration of the 9GHz e.p.r. (electron-paramagnetic-resonance) spectrum (Wyard, 1965), suggesting there are two e.p.r.-detectable Cu(II) ions/molecule of enzyme. These results are in general agreement with previous studies (Buffoni et al., 1968). E.p.r. spectra on the enzyme at 9 and 36GHz have been computer-simulated and indicate that the two Cu(I1) ions are in different chemical environments of close-to-axial symmetry. At present it is not possible to distinguish whether the two Cu(I1) ions are associated with the two subunits or are located at the subunit interface, To further characterize the ligand environment of the copper atoms, proton relaxation times (TI and T2) have been measured as a function of frequency (10, 30 and 6OMHz) and temperature (273-323°K) for benzylamine oxidase (0.5 mM) in 5 mwphosphate buffer, pH7.0. The relaxation rates (1/T) were found to be between 10- and 50-fold greater than in the buffer alone. These results indicate that water molecules co-ordinated axially to the Cu(I1) ions are exchanging rapidly with water molecules in the bulk aqueous phase. In contrast, similar measurements on a solution of the copper protein azurin have shown that the Cu(I1) ion site is not accessible to the water molecules from bulk solution. In principle it should be possible to determine from the relaxation-rate measurements the number of co-ordinated water molecules, their exchange rates and their distance from the Cu(II) ion (Peacocke et al., 1969). However, this approach, which has been reasonably successful with Mn(1I) enzymes, does not yield realistic values for these quantities .