Kinetic studies on the oxidation of phenols by the horseradish peroxidase compound II

Oxidation of substituted phenols by horseradish peroxidase compound II were studied using stopped-flow technique. Dissociation constants (K(D)) of HRP-II-phenol complexes were deduced from the kinetic data. Magnitudes of K(D) fall in a relatively narrow range of 3-11 mM. These are comparable to 3-10 mM reported for the binding of substituted phenols to native HRP, suggesting that the mode of binding of phenols to native HRP and HRP compound II may be similar. pH dependence of the apparent second order rate constants (k(app)) of the reactions of all the phenols were determined. The k(app) values of reactions other than the reaction of tyrosine, were observed to increase in the acidic region but decreased in the alkaline region. The increase was attributed to the deprotonation of distal carboxylic acid residue on enzyme with pK(a) values of 4.2-5.2. For tyrosine, however, the apparent second-order rate constant was observed to further increase non linearly on increasing the pH in the alkaline region. Results were interpreted quantitatively on the basis that protonated form of the enzyme reacted with the protonated form of the phenol with different individual rate constants.