Kinetic characterization of a fungal peroxidase from Coprinus cinereus in aqueous and organic media

Catalytic and structural characteristics of a fungal peroxidase from Coprinus cinereus (CiP) expressed by Aspergillus niger were investigated in aqueous media and aqueous/dioxane solvents, and compared to the well-known peroxidase from horseradish (HRP). CiP is more thermostable than HRP; the half lives for the irreversible inactivation of CiP and HRP at 85 degrees C were 40 min and 21 min, respectively. The observed oxidation potential of CIP is similar to that of HRP. The kinetics for the oxidation of substituted phenols catalyzed by both peroxidases were measured to construct linear free energy relationships between the catalytic efficiency, V-max/K-m, of both enzymes and the electronic and hydrophobic properties of the phenolic substrates as represented by the Hammett constant, sigma and Hansch constant, pi, respectively. CiP and HRP are similar in response to substrate hydrophobicity in different concentrations of dioxane; a finding not unexpected as substrate desolvation dominates hydrophobic interactions in organic media. The two peroxidases are quite dissimilar, however, in response to the electronic property of the substrate. In aqueous buffer, CiP is less sensitive than HRP to the electron donating/withdrawing properties of the phenolic substrates and shows a Hammett coefficient, rho, of -0.66. As the dioxane content increased, however, CiP becomes more sensitive to the electronic properties of the phenolic substrates with a rho value of - 0.38 in 80% v/v dioxane. The dependence of rho on the dioxane content suggests that the active site of CiP is exposed to the reaction medium and this has a strong influence on the substrate specificity of this fungal enzyme.