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 pKa 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 (EH‡2O), indicating that the enthalpy change associated with the binding of MnHRP to H2O2 may be positive.