Reactions of bovine serum amine oxidase with NN-diethyldithiocarbamate. Selective removal of one copper ion.
NN-Diethyldithiocarbamate (DDC) was able to bind, at 1.0 mM concentration, only about 50% the Cu(II) ions of bovine plasma amine oxidase. Under reducing conditions, this Cu(II) was removed with inactivation of the enzyme. Up to 90% activity could be recovered by treatment with excess Cu(II). The organic cofactor, sensitive to carbonyl reagents, was reduced in the half-Cu-depleted protein and no longer bound phenylhydrazine. The fully reacted protein, in the presence of 10 mM-DDC, lost 50% Cu(II) upon storage at -20 degrees C, but in this case the residual Cu(II) was in the DDC-bound form and the cofactor was in the oxidized state, as it could still bind phenylhydrazine. In the presence of DDC, the rate of reaction with phenylhydrazine was always low, even at 50% DDC saturation, and all derivatives showed identical modifications of the optical and e.p.r. spectra with respect to the phenylhydrazone of the native protein. It is concluded that the two Cu(II) ions are not equivalent, that removal of a single Cu(II) is sufficient to inhibit the re-oxidation of the organic cofactor, and that both Cu(II) ions are in some way involved in the reaction with phenylhydrazine. After reaction with DDC, the optical and e.p.r. spectra of 63Cu(II)-amine oxidase and of 63Cu(II)-carbonic anhydrase [Morpurgo, Desideri, Rigo, Viglino & Rotilio (1983) Biochim. Biophys. Acta 746, 168-175] are very similar and show distorted equatorial co-ordination to Cu(II) of two sulphur atoms and two magnetically equivalent nitrogen atoms.
Morpurgo
L
L
Dipartimento di Scienze Biochimiche, Università di Roma La Sapienza, Italy.
Agostinelli
E
E
Befani
O
O
Mondovì
B
B
D006631:Amine Oxidase (Copper-Containing); D000818:Animals; D001051:Apoenzymes / Q000097:blood; D001665:Binding Sites; D002256:Carbonic Anhydrases; D002417:Cattle; D003300:Copper / Q000378:metabolism*; D004050:Ditiocarb / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D010084:Oxidation-Reduction; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000097:blood*; D013053:Spectrophotometry
D001051:Apoenzymes; D003300:Copper; D004050:Ditiocarb; D006631:Amine Oxidase (Copper-Containing); D000587:Oxidoreductases Acting on CH-NH Group Donors; D002256:Carbonic Anhydrases