Kinetic studies of the reaction of hydrogen peroxide with manganese-reconstituted horseradish peroxidase

Abstract

The kinetics of the transient reaction of hydrogen peroxide with manganese-reconstituted horseradish peroxidase (MnHRP) has been studied using stopped-flow spectrophotometry. The specificity of the reaction seems to be maximal at physiological pH. The bell-shaped pH dependence of the formation of the resulting ‘peroxide’ compound I has been interpreted in terms of two ionisable groups at the active site of the enzyme; the pK_a of one group was found to be near 4.8 and that of the other near 10.6. Values of the apparent second-order rate constant determined at various temperatures in the range 20–50 °C were used to calculate the thermodynamic parameters of the reaction. The apparent activation energy for the formation of MnHRP compound I was found to be higher than that of the native peroxidase and the value of the minimum energy of activation in water (E_H^‡2O), indicating that the enthalpy change associated with the binding of MnHRP to H₂O₂ may be positive.