KINETIC-STUDIES OF THE REACTION OF HYDROGEN-PEROXIDE WITH MANGANESE-RECONSTITUTED HORSERADISH-PEROXIDASE

The kinetics of the transient reaction of hydrogen peroxide with manga nese-reconstituted horseradish peroxidase (MnHRP) has been studied usi ng stopped-flow spectrophotometry. The specificity of the reaction see ms to be maximal at physiological pH. The bell-shaped pH dependence of the formation of the resulting 'peroxide' compound I has been interpr eted 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 othe r near 10.6. Values of the apparent second-order rate constant determi ned at various temperatures in the range 20-50 degrees C were used to calculate the thermodynamic parameters of the reaction. The apparent a ctivation energy for the formation of MnHRP compound I was found to be higher than that of the native peroxidase and the value of the minimu m energy of activation in water (E(H2O)(double dagger)), indicating th at the enthalpy change associated with the binding of MnHRP to H2O2 ma y be positive.