Phenolic and non phenolic substrates oxidation by laccase at variable oxygen concentrations: Selection of bisubstrate kinetic models from polarographic data
Abstract Using laccase as a case study, an alternative methodology for discrimination of bisubstrate mechanism types, useful for Bi Bi reactions when water is one of the products, is presented. A purified enzyme from fungal cultures of Ganoderma resinaceum and two reducing substrates: 2,2' azino bis(3 ethylbenzthiazoline 6 sulphonic acid) (ABTS) and 2,6 dimethoxyphenol (DMP) are used. Kinetic assays of ABTS or DMP oxidation using variable initial concentrations of dioxygen are performed in a closed system and depletion rates of dioxygen are measured using polarography. Seven bisubstrate models based on Michaelis Menten Bi Bi mechanisms are applied to the experimental data. Akaike information criterion, a penalized likelihood analysis, and Akaike weights are used for model selection. Results show that the more likely bisubstrate models for ABTS and DMP oxidation are ping pong and Theorell Chance, respectively. However, the catalytic efficiency of oxygen reduction to water exhibits similar values, and is compatible with a process relatively independent of reducing substrates and the bisubstrate mechanism type. Finally, Michaelis Menten constants towards dioxygen, 233 mM with ABTS and 700 mM with DMP, indicate that the soluble electron acceptor available for laccase catalyzed reactions, is far removed from saturation.