Abstract
A novel copper-depleted bovine serum amine oxidase (BSAO), in which about half the molecules contained the organic cofactor in the oxidized form, was prepared by adding a reductant in anaerobic conditions to the cyanide-reacted protein. The CuI-semiquinone formed in these conditions reoxidizes after the removal of copper. The inactive derivative was reduced by benzylamine at approx. 1/1000 the rate of BSAO. The pseudo-first-order reaction was preceded by the formation of a protein-benzylamine complex with dissociation constant, Kd, of 4.9+/-0.5 mM, similar to the Km of BSAO (2.2 mM). Also the reactions with phenylhydrazine and benzohydrazide were considerably slower than in holo-BSAO, whereas the reactions with p-pyridine-2-ylphenylacetohydrazide, containing a longer aromatic tail, and semicarbazide, lacking an aromatic moiety, were less severely affected. Removal of copper had no effect on the optical spectra of BSAO and of most adducts, containing the cofactor in quinol form, showing that copper is bound to neither the oxidized nor the reduced cofactor. Benzylhydrazine did not produce optical effects but was tightly bound, as inferred from its inhibitory effect on reaction with other molecules. Substrate and inhibitors might bind a hydrophobic pocket at some distance from the quinone, probably near the protein surface, with their affinity depending on the hydrophobic character and pKa. The binding, which is not greatly influenced by copper removal, probably induces a copper-dependent change of conformation, 'opening' a pathway to the active site buried in the protein interior.