Galactose oxidase general

Fig. 21. Proposed catalytic mechanism for substrate oxidation by galactose oxidase. (A) Substrate binding displaces Tyr-495 phenolate which serves as a general base for abstracting the hydroxylic proton. (B) Stererospecihc pro- hydrogen abstraction by the Tyr-Cys phenoxyl radical. (C) Inner sphere electron transfer reducing Cu(II) to Cu(I). (D) Dissociation of the aldehyde product.

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. 

Since both alcoholic oxidation and O2 reduction are two-electron processes, the catalytic reaction is conceptually equivalent to a transfer of the elements of dihydrogen between the two substrates. Biological hydrogen transfer generally involves specialized organic redox factors (e.g., flavins, nicotinamide, quinones), with well-characterized reaction mechanisms. Galactose oxidase does not contain any of these conventional redox factors and instead utilizes a very different type of active site, a free radical-coupled copper complex, to perform this chemistry. The new type of active site structure implies that the reaction follows a novel biochemical redox mechanisms based on free radicals and the two-electron reactivity of the metalloradical complex. 

Copper would seem to be an appropriate choice of metal for the catalytic oxidation of alcohols with dioxygen since it comprises the catalytic centre in a variety of enzymes, e.g. galactose oxidase, which catalyze this conversion in vivo However, despite extensive efforts synthetically useful copper-based systems have generally not been forthcoming. 

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