Roles of the two copper ions in bovine serum amine oxidase

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Abstract
With a view to obtaining information on the roles of the two copper ions in bovine serum amine oxidase (BSAO), spectroscopic and magnetic studies on several BSAO derivatives have been carried out. Cu-depleted BSAO (Cu-depBSAO) exhibits no enzyme activity and only a few a low absorption intensity at ca. 475 nm, which is the characteristic absorption maximum of the chromophore in BSAO. The binding of 1 mol of Cu to 1 mol of Cu-depBSAO slightly but definitely increases the enzyme activity and the absorptivity, although they are much lower than those of native BSAO. The incorporation of 2 mol of Cu into Cu-depBSAO gives rise to a similar high activity and absorptivity as those of the native enzyme. Electron paramagnetic resonance (EPR) spectra of the BSAO derivatives reveal that two copper ions in the enzyme molecule are environmentally identical. Titrations of BSAO, Cu-depBSAO, and Cu-half-depleted BSAO (Cu-half-depBSAO), containing 1 mol of copper per mole of protein, with phenylhydrazine (an inhibitor of BSAO) indicate that only 1 mol of phenylhydrazine reacts with 1 mol of the enzyme. In other words the enzyme possesses only one chromophore or one active site, though the molecule is composed of two electrophoretically dientical subunits. The binding constants between phenylhydrazine and BSAO, Cu-depBSAO, or Cu-half-depBSAO were estimated to be 5 .times. 106, 5 .times. 104, and 1 .times. 105 M-1, respectively. The binding of phenylhydrazine to the chromophore is assisted by the presence of two copper ions by a factor of 100. One of the most important roles of copper ions in BSAO is likely to retain the chromophore in a favorable geometric and electronic structure for binding the substrate.