Copper in galactose oxidase

Given some notion of the nature of the endogenous ligands, we can next ask how exogenous ligands perturb the copper atom and what this can tell us about the electronic transitions exhibited by the Cu(II) atom in galactose oxidase. 

Table III). Atomic absorption analysis shows that the copper is still present in the enzyme. These two facts coupled with the 600-nm region intensity increase might be interpreted to suggest that a stereochemical distortion accompanies tryptophan oxidation in galactose oxidase. For example, a saddling of the planar environment would be expected to lower the reduction potential of the Cu(II)-Cu(I) couple. 

Oxidation of this tryptophan in galactose oxidase also prevents alkylation of the histidine residue. Alkylation of the histidine residue in turn markedly affects the fluorescence quantum yield of this tryptophan (43) and nearly abolishes the absorbance of the copper atom. The copper atom itself is also essential to the reactivity of this histidine. Thus, we appear to have a consistent set of highly interdependent components. Not unexpectedly, the copper site cannot be fully understood without considering its interactions with non-ligand protein groups. 

Figure 3. Sketch of the copper coordination in galactose oxidase, as determined by the 1.7 A resolution crystal structure.

The alcohol dehydrogenases were already described in Chapter 3. These enzymes are cofactor dependent and in the active site hydrogen transfer takes place from NADH or NADPH. In the reverse way they can, however, be applied for the oxidation of alcohols in some cases (see below). Oxidases are very appealing for biocatalytic purposes, because they use oxygen as the only oxidant without the need for a cofactor. Oxidases usually have flavins (glucose oxidase, alcohol oxidase) or copper (examples galactose oxidase, laccase and tyrosinase) in the active site [18]. The mechanism for glucose oxidase (GOD) is denoted in... 

Fig. 8. Tyrosine coordination modes in galactose oxidase-copper complex. Tyrosine phenolate bond angles (0) and ring torsion angles (t) are indicated. (Based on protein coordinates PDB ID IGOG.)...

Whittaker, J. W., 1994, The free radical-coupled copper active site in galactose oxidase. Metal Ions in Biology, Editors Sigel, H. and Sigel, A., Marcel Dekker publishers, 30 3159 360. 

Use of apoenzymes for the detection of metal ions Generally, apoenzymes of metalloenzymes can be used for the detection of the corresponding metal ion. Restoration of enzyme activity obtained in the presence of the metal ion can be correlated to its concentration. This principle has been demonstrated in the detection of copper while evaluating reconstituted catalytic activities in galactose oxidase and ascorbate oxidase and also in the detection of zinc since this ion is essential for the activity of carbonic anhydrase and alkaline phosphatase [416]. The need of stripping the metal for the preparation of the apoenz5une may demand tedious procedures and a catalytic assay with the addition of the substrate is always required for detection. 

The oxidation state of copper in biological systems is -1-1 or +2. Copper(III) is found in inorganic systems and may occur as a reaction intermediate in galactose oxidase, laccase (a plant enzyme), and perhaps other enzymes. The coordination number of copper in these enzymes ranges from two to six and occasionally higher. 

Type 2 copper centers are not uniform in ligand or ligand stereochemistries. One common feature is, however, that in the active enzyme, one coordination site is always free to bind oxygen. The most common ligand in type 2 copper centers is histidine. Tyrosine (often modified), methionine, and cysteine occur as well. There are three histidines and a modified tyrosine in amine oxidase and lysyl oxidase [28]. In diamine oxidase, two of the histidine residues have probably been replaced by cysteines [29]. In galactose oxidase, the copper ion is coordinated by two tyrosines, two histidines and an acetate ion [30]. Dopamine-/J-hydroxylase contains two differently coordinated copper ions per functional unit. One is coordinated by three histidines and a methionine and the other by two histidines and another, yet unknown, ligand [ 31 ]. Last but not least, the type 2 copper ion in Cu,Zn-superoxide dismutase is coordinated by four histidine residues, one of which connects the copper ion to the zinc ion, the second metal ion in the active site of the enzyme [32,33] (Fig. 6). 

The copper center. Galactose oxidase is capable of catalyzing a two-electron reduction, although it only contains a single type 2 copper center. This assumption is, however, incorrect. Recent studies show that the copper ion occurs in its divalent state, Cu2+, which is antiferromagnetically coupled to a tyrosyl radical (Tyr272) and is, therefore, ESR-inactive [158]. 

Jazdzewski BA, Tolma WB (2000) Understanding the copper-phenoxyl radical array in galactose oxidase contributions from synthetic modeling studies. Coord Chem Rev 200-202 633-685... 

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