O-Galactose oxidase

The enzyim o-galactose oxidase has been reported to oxidize some racemic diols with kinetic resolution to provide the corresponding hydroxy aldehydes with high enantiomeric excesses. A list of successful and unsuccessful substrates is given in Scheme 4. 

Glycoproteins have been located on the surface of two murine leukaemic cells, as shown by o-galactose oxidase-sodium borotridite and I-lacto-peroxidase-iodination techniques. The patterns on polyacrylamide gel electrophoresis of the solubilized glycoproteins from the two sources are indistinguishable but differ from those of the surface glycoproteins of HeLa cells. 

So-called blue multinuclear copper oxidase enzymes, such as laccase and ascorbate oxidase, catalyze the stepwise oxidation of organic substrates (most likely in successive one-electron steps) in tandem with the four-electron reduction of O2 to water, i.e. no oxygen atom(s) from O2 are incorporated into the substrate (Eq. 4) [15]. Catechol oxidase, containing a type 3 center, mediates a two-electron substrate oxidation (o-diphenols to o-chinones), and turnover of two substrate molecules is coupled to the reduction of O2 to water [34,35]. The non-blue copper oxidases, e.g. galactose oxidase and amine oxidases [27,56-59], perform similar oxidation catalysis at a mononuclear type 2 Cu site, but H2O2 is produced from O2 instead of H2O, in a two-electron reduction. 

Galactose oxidase of P. circinatus was apparently inhibited by traces of BESOD. It can be inactivated by H Oj produced in the reaction unless catalase was added. There was an activation by traces of O J. In the absence of oxidants the reaction usually showed an induction period The enzyme, used at very low concentrations in the assays, was protected by proteins like serumalbumin. SOD did, however, not alter the reaction rate when added after 15 min This was interpreted by an inactivation of SOD by the H O accumulated in the reaction but it could just as well mean that SOD had no effect on the active enzyme, but that it did lower the activation in the induction period. Peroxidase activated galactose oxidase and suppressed the effect of SOD It did protect the enzyme against H O inactivation and could have been responsible for appreciable amounts of OJ, produced from O and from radicals formed in its action on a substrate. 

There is no valid interpretation for the activation by OJ and by hexacyano-ferrate(III), although they fitted nicely in a reaction scheme with Cu(III) as the active species In the oxidation of an alcohol to an aldehyde Cu(III) would be reduced to Cu(I). In the subsequent reaction of Cu(I) with Oj, Cu(II)Oj was considered an intermediate yielding Cu(III) and H O. This intermediate would be in a reversible equilibrium with OJ and with the resting Cu(II)-enzyme. This resting enzyme would be oxidized by hexacyanoferrate(III) to the active Cu(III) species. There was unfortunately no indication in X-ray absorption measurements for the formation of Cu(III) with hexacyanoferrate(III) and the resting enzyme . EPR measurements indicated that Cu(II) was present in the active enzyme It was not possible, moreover, to detect Oj by the reduction of Fe(III)-cytochrome c in a galactose oxidase — galactose system... 

Newer assays involve the action of enzymes, e.g., the assay of Baird and Smith (1989b) who treated galactomannans with galactose oxidase to generate H202 that in turn oxidized o-tolidine in the presence of preoxidase. These authors claimed this double oxidation reaction to be specific for the galactosyl monomer and its derivatives, based on the exclusive oxidation at the C-6 position. o-Phenylenediamine can be substituted for o-tolidine the color is measured at 425 nm. 

T17. Tressel, R, and Kosman, D. J., o,o-Dityrosine in native and horseradish peroxidase-activated galactose oxidase. Biochem. Biophys. Res. Commun. 92, 781-786 (1980). 

Babcock, G. T., El-Deeb, M. K., Sandusky, P. O., Whittaker, M. M., and Whittaker, J. W., 1992, Electron paramagnetic resonance and electron nuclear double resonance spectroscopies of the radical site in galactose oxidase and of thioether-substituted phenol model compounds, J. Am. Chem. Soc. 114 372793734. 

Wang, Y., DuBois, J. L., Hedman, B., Hodgson, K. O., and Stack, T. D. P., 1998, Catalytic Galactose Oxidase Models Biomimetic Cu(II)-Phenoxyl-Radical Reactivity, Science 279 5379540. 

As a mimic of the well-studied galactose oxidase [37], a copper(II) thiophenol complex catalyzes the oxidation of primary alcohols to aldehydes in the presence of (Scheme 12) [38]. The latter also promotes the oxidation of secondary alcohols to diols (Scheme 12). The catalytic cycle starts with the oxidation of copper by O, leading to a biradical species. The intermediate 39 is produced from 38 by coordination of two alkoxide substrates. The rate-limiting step is the formation of 40 from 39 by a hydrogen atom transfer from the secondary alcoholate to the oxygen-centered radicals of the aminophenols ligands. The cycle is then closed by radical dimerization which leads the formation of the diol [39]. 

Galactose oxidase operates by a free radical mechanism. The enzyme is isolated as a mixed redox state in which only some 5% of molecules are active. Full activation can be achieved in vitro by a number of one-electron oxidants that facilitate the removal of one electron to create the Tyr272-based radical (Figure 16, R O). 

Many other metalloenzymes with associated or coupled ligand radicals are known. Himo el have studied the catalytic cycle of galactose oxidase where a Cu center is spin coupled to a bound tyrosine radical in the substrate binding state. After proton transfer to the Tyr-O in the first step of the catalytic cycle, the radical center moves to the Cu bound tyrosine which is cross-linked to cysteine-S. In subsequent steps of the reaction, the ligand radical is propagated to the substrate, and the Cu is reduced to Cu by e transfer. Then, a reaction with O2 regenerates the Cu -tyrosine radical starting state. 

Specificity for the alcohol substrate is very broad, ranging from small molecules (e.g. propanediol) to polysaccharides. Galactose oxidase is strictly stereo specific. It does not oxidise either o-glucose or l-galactose. 

Agarose.—Macroporous agarose has been used in the purification of D-galactose oxidase and of o-galactose-binding lectins from castor beans Ricinus communisf by affinity chromatography. Microscopic examination has shown that beaded... 

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