Ceruloplasmin substrates

A controversial function of ceruloplasmin in vivo is its ability to oxidize ferrous to ferric iron as a substrate Fe(II) has the lowest apparent Km and the highest Vmax of any of the protein s multiple substrates. 

The multi-copper oxidases include laccase, ceruloplasmin, and ascorbate oxidase. Laccase can be found in tree sap and in fungi ascorbate oxidase, in cucumber and related plants and ceruloplasmin, in vertebrate blood serum. Laccases catalyze oxidation of phenolic compounds to radicals with a concomitant 4e reduction of O2 to water, and it is thought that this process may be important in the breakdown of lignin. Ceruloplasmin, whose real biological function is either quite varied or unknown, also catalyzes oxidation of a variety of substrates, again via a 4e reduction of O2 to water. Ferroxidase activity has been demonstrated for it, as has SOD activity. Ascorbate oxidase catalyzes the oxidation of ascorbate, again via a 4e reduction of O2 to water. Excellent reviews of these three systems can be found in Volume 111 of Copper Proteins and Copper Enzymes (Lontie, 1984). 

The blue oxidases contain these three types of copper together The stoichiometry is straightforward with laccase which contains one type-1 and one type-2 copper, and one type-3 dimeric copper site . One would expect two laccase-like sites in ascorbate oxidase and in ceruloplasmin, but the presence of respectively 3 and 1 and 1 and 3 type-1 and type-2 copper atoms has been deduced. Ceruloplasmin shows oxidase activities towards different substrates, like Fe (ferroxidase) and aromatic amines. It plays, moreover, an active role in the transport of copper With the proper precautions against the action of proteinases it can be isolated as a single polypeptide chain... 

This blue oxidase, present in the plasma of vertebrates, appears to be multifunctional.905,975 It accounts for some 95% of the circulating copper in a normal mammal, and its concentration fluctuates considerably in diseased states. It appears that ceruloplasmin has a major role in copper transport (as discussed in Section 62.1.11). In addition it has oxidase activity towards three groups of substrates, although its physiological role is not known with certainty. 

This is directed particularly towards Fe11,976 the substrate with the highest Vm and the lowest Km values. Ceruloplasmin-catalyzed oxidation of Fe is 10-100 times faster than the non-enzymatic reaction, and ceruloplasmin appears to be the only effective ferrooxidase in human serum. This is linked to the control of iron mobilization by ceruloplasmin. Iron is released as Fe from ferritin, and the rate at which it is converted to circulating Fem2-transferrin is dependent upon ceruloplasmin. This explains why Cu-deficient animals develop anaemia. 

The several functions of ceruloplasmin cannot be explained at present. It seems reasonable that this diversity is related to the activity of the copper centres. The general pattern of oxidase activity is probably similar to that of the other blue oxidases, with a type 3 binuclear site serving to bind and reduce dioxygen, with electrons transferred from the type 1 site. The type 2 copper may represent a substrate-binding site. 

In the discussion of the biochemistry of copper in Section 62.1.8 it was noted that three types of copper exist in copper enzymes. These are type 1 ( blue copper centres) type 2 ( normal copper centres) and type 3 (which occur as coupled pairs). All three classes are present in the blue copper oxidases laccase, ascorbate oxidase and ceruloplasmin. Laccase contains four copper ions per molecule, and the other two contain eight copper ions per molecule. In all cases oxidation of substrate is linked to the four-electron reduction of dioxygen to water. Unlike cytochrome oxidase, these are water-soluble enzymes, and so are convenient systems for studying the problems of multielectron redox reactions. The type 3 pair of copper centres constitutes the 02-reducing sites in these enzymes, and provides a two-electron pathway to peroxide, bypassing the formation of superoxide. Laccase also contains one type 1 and one type 2 centre. While ascorbate oxidase contains eight copper ions per molecule, so far ESR and analysis data have led to the identification of type 1 (two), type 2 (two) and type 3 (four) copper centres. 

Blue Multicopper Oxidases. These include laccases, ascorbate oxidase, and ceruloplasmin [22,61], which along with cytochrome c oxidase (CcO with Fe and Cu) can couple the one-electron oxidation of substrates (e.g., ascorbate, diamines, monophenols Fe2+ for ceruloplasmin cytochrome c, for CcO) to the full reduction of dioxygen to water (i.e., 02 + 4c + H+ —> 2H20). 

The enzyme catalyses the oxidation of L-ascorbic acid, 4, to dehydroascorbic, 5. The blue protein belongs to the group of blue oxidases together with laccase and ceruloplasmin. These are multicopper enzymes catalysing the electron reduction of molecular oxygen to water with concomitant one electron oxidation of the substrate. 

According to the Chance mechanism, the interaction of H202 with the enzyme gives compound I (E,). The oxidation of the donor molecules leads to compound II (E[I) which oxidizes the second donor molecule. The radical intermediates were detected experimentally for such substrates as amines and phenols with relatively high reduction potential (Dunford and Stillman, 1976). The one-electron steps with the formation of free radicals at oxidation of amines and phenols have been proved in the ceruloplasmin, laccase and ascorbic oxidase reactions (Malsmstrom et al., 1975). 

An important family of multicopper enzymes couple the reduction of O2 to H2O with substrate oxidation. They include ascorbate oxidase, ceruloplasmin, Fet3, hephaestin, and laccase, and contain at least four copper ions. The four Cu ions are distributed between one type 1 blue copper site, one type 2 site, and one type 3 copper site. The blue Type 1 site is usually located some 12—13 A distant from a trinuclear site which has the two Type 3 coppers, linked by a bridging oxygen and one Type 2 copper. We illustrate this class of oxidases with laccase which catalyses the four-electron reduction of O2 to water, coupled with the oxidation of small organic... 

Ceruloplasmin is an enzyme exhibiting oxidase activity against several substrates with a pH optimum between 5.4 and 5.9. The best known substrate is p-phenylenediamine or its dimethyl derivative. The oxidase activity is much weaker against other substrates such as hydroquinone, catechol, pyrogallol, DOPA, adrenaline, noradrenaline, serotonin (L4). The physiological substrate of ceruloplasmin, if any, has not yet been found. 

The blue oxidases like ascorbate oxidase, laccase, and ceruloplasmin, and the terminal oxidases of aerobic respiratory chains like cytochrome oxidases and quinol oxidases are the only enzymes so far known that catalyze the direct four-electron reduction of molecular oxygen to water. Thereby, the reducing substrates like ascorbate, quinol, Fe " ", and cytochrome c are oxidized in one-electron transfer steps. The substrates of quinol oxidases, ubiquinol, or menaquinol, may be oxidized in two-electron transfer steps. For the two cases the following general reaction formulae can be defined ... 

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