STRUCTURE-FUNCTION STUDIES OF HUMAN CERULOPLASMIN
V.Zaitsev1, I.Zaitseva1,
G.Card2 & P.Lindley3
1CCLRC Daresbury
Laboratory, Warrington WA4 4AD,UK
2Imperial Cancer
Research Fund, 44 Lincoln's Inn Fields,London WC2A 3PX,UK
3Eropean Synchrotron
Radiation Facility (ESRF), B.P.220 38043 Grenoble Cedex-France
Human plasma ceruloplasmin (hCP) is a copper-containing glycoprotein with a molecular weight of 132 kDa. It belongs to a family of multinuclear "blue" oxidases along with ascorbate oxidase and laccase and it is comprised of a single chain of 1046 amino acids with a carbohydrate content of 7% to 8% (1). The precise functions of CP have not been defined, but it has been suggested a multifunctional role for the enzyme in the blood including a ferroxidase, amine oxidase, antioxidant activities and copper transport (2,3). hCP catalyzed oxidation of biogenic amines may be of importance in regulating the level of the stress hormones in the bloodstream and eventually in those areas of the brain where these substances act as neurotransmitters. The effects of drugs used in the treatment of mental illness, e.g., tranquilizers and antidepressants on oxidation of biogenic amines by hCP have been studied (4 ).
The X-Ray structure (5) reveals that the hCP molecule is comprised of six cupredoxin type domains with large loop insertions. There are six integral copper atoms, three of which form a trinuclear cluster sited at the interface of domain 1 and 6 and mononuclear sites in domains 2, 4 and 6. A series of soaking and co-crystallisation experiments has been carried out under a variety of conditions to attemt to characterize the chemistry of binding of anions, cations and organic inhibitors and substrates. Two binding sites of Fe(II) substrate have been identified in domains 4 and 6. Possible electron transfer pathways from ferrous ions through the labile copper sites to the mononuclear coppers and trinuclear cluster have been suggested (6). The ferroxidase role of hCP in plasma may thus be substantiated, whereby hCP is involved in mediating the release of iron from cells, oxidation of the iron to the Fe(III) state, and its subsequent incorporation into apo-transferrin. Further X-ray studies have been undertaken involving complexes with azide and organic substrates: p-phenylenediamine, biogenic amines (adrenaline, noradrenaline, serotonine, dopa), d-lysergic acid diethylamide (LSD) and Cl-promazin. The binding sites for these compounds have been identified on the difference Fourier maps. The results of these experiments will be discussed from which mechanisms of the substrate oxidation can be hypothesised