Horseradish peroxidase. XXVIII. Formation and reactivity of the alkaline form. Evidence for an enzyme–substrate complex in compound 1 formation

Abstract

The rate of formation of 1 from horseradish peroxidase and hydrogen peroxide is investigated as a function of pH between pH9.5 and 11.5. For pH values smaller than 10, the rate depends linearly on hydrogen peroxide concentration, whereas between pH 10 and 11.5, the rate displays saturation kinetics. Evidence is presented for the formation of an intermediate complex before compound 1 formation. Two parameters were measured as a function of pH: kapp, the apparent rate constant for compound 1 formation, and kapp, the dissociation rate constant of the intermediate complex to 1. A third parameter, kapp, similar to a dissociation constant for the reaction was deduced from the two rate constants. The apparent rate constant kapp is pH independent for pH values < 9.5 and is directly affected by ionization of only one group on the native enzyme with a pK of 11. The pH dependances of kd and Kapp require a minimum of two ionizations. One corresponds to a group on the native enzyme which ionizes at a pH lower than that of the present study, the second corresponds to the ionizable group with pK of 11. For cyanide binding to horseradish peroxidase over the same pH interval, saturation kinetics were not observed, yet the pH profile for kapp exactly parallels that for formation of 1 (when corrected for the effect of the ionization of HCN) and shows an inflection at the same pH value.