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

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Abstract
The rate of formation of compound 1 from horseradish peroxidase and hydrogen peroxide is investigated as a function of pH between pH 9.5 and 11.5. For pH values smaller than 10, the rate depends linearly on H2O2 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 kd, 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 2 rate constants. The apparent rate constant kapp is pH independent for pH values < 9.5 and is directly affected by ionization of only 1 group on the native enzyme with a pK of 11. The pH dependences of kd and Kapp require a minimum of 2 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.

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