Copper removal from pig kidney amine oxidase containing Cu/topaquinone (TPQ) has been obtained using CN in the presence of the poor substrate p-(dimethylamino)benzylamine. Upon removal of copper, the enzyme loses its activity while the TPQ cofactor remains in its oxidized form. The addition of copper to the apo-form fully restores the active enzyme. The CN treatment in the presence of sodium dithionite or good substrates (cadaverine or benzylamine) also removes copper but the TPQ cofactor is irreversibly reduced and the addition of copper does not regenerate the active enzyme. NiII and ZnII do not bind the apo-protein in contrast to CoII which is incorporated to the same extent as CuII. However, Co-reconstituted enzyme only shows a very low activity. These results demonstrate that copper is essential for the catalytic mechanism because it maintains the correct active site geometry.
Properties of copper-free pig kidney amine oxidase: Role of topa quinone
PADIGLIA, ALESSANDRA;MEDDA, ROSARIA;PINTUS, FRANCESCA;
2006-01-01
Abstract
Copper removal from pig kidney amine oxidase containing Cu/topaquinone (TPQ) has been obtained using CN in the presence of the poor substrate p-(dimethylamino)benzylamine. Upon removal of copper, the enzyme loses its activity while the TPQ cofactor remains in its oxidized form. The addition of copper to the apo-form fully restores the active enzyme. The CN treatment in the presence of sodium dithionite or good substrates (cadaverine or benzylamine) also removes copper but the TPQ cofactor is irreversibly reduced and the addition of copper does not regenerate the active enzyme. NiII and ZnII do not bind the apo-protein in contrast to CoII which is incorporated to the same extent as CuII. However, Co-reconstituted enzyme only shows a very low activity. These results demonstrate that copper is essential for the catalytic mechanism because it maintains the correct active site geometry.
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