Substrate specificity of a purified fungal laccase

The substrate specificity of a pure laccase preparation from the fungus Polyporus versicolor has been studied. A wide range of substrates were readily oxidized, among them catechol, hydroquinone, guaiacol and guaiacyl derivatives, resorcinol, and phloroglucinol. The oxidation of p-cresol was studied in particular detail. The experiments showed that p-cresol was oxidized very incompletely in a system containing substrate, buffer and the pure enzyme. In the presence of catechol, both substances were oxidized at a comparatively rapid rate and with an oxygen uptake which could be expected stoichiometrically. This was first interpreted as an indirect oxidation of p-cresol via intermediary oxidation products of catechol. Later experiments speak, however, in favour of the explanation that p-cresol is directly oxidized but that it rapidly inactivates the laccase and that catechol exerts a protective influence. Gelatin and Tween 80 proved to be still more efficient protective agents. In the presence of Fe++, but not Mn++, the oxidation of p-cresol was also stimulated. Similar results were obtained with hydroquinone monoethyl ether and α-naphthol, which both inactivate laccase at low concentrations. Certain guaiacyl derivatives show a slighter inhibition, but a detailed investigation of these substances has not been made. The diphenols catechol and hydroquinone do not inactivate laccase. There are certain similarities with tyrosinase insofar that the “cresolase” activity of this enzyme is more easily destroyed than the “catecholase” activity. The results suggest that the monophenolase and polyphenolase activities occupy different sites on the laccase molecule. The monophenolase site is more unstable than the polyphenolase site.