Kinetics of formation of the primary compound (compound I) from hydrogen peroxide and turnip peroxidases

Abstract

A kinetic study of the reaction of two turnip peroxidases (P1 and P7) with hydrogen peroxide to form the primary oxidized compound (compound I) has been carried out over the pH range from 2,4 to 10.8. In the neutral and acidic pH regions, the rates depend linearly on hydrogen peroxide concentration whereas at alkaline pH values the rates display saturation kinetics. A comparison is made with the cyanide binding reaction to peroxidases since the two reactions are influenced in the same manner by ionization of groups on the native enzymes. Two different ionization processes of peroxidase P1 with pKa values of 3.9 and 10 are required to explain the rate pH profile for the reaction with H2O2. Protonation of the former group and ionization of the latter causes a decrease in the rate of reaction of the enzyme with H2O2. In the case of peroxidase P7 a minimum model involves three ionizable groups with pKa values of 2.5, 4, and 9. Protonation of the former two groups and ionization of the latter lowers the reaction rate. In the pH-independent region, the rate of formation of compound I was measured as a function of temperature. From the Arrhenius plots the activation energy for the reaction was calculated to be 2.9 ± 0.1 kcal/mol for P1 and 5.4 ± 0.3 kcal/mol for P7. However, the rates are independent of viscosity in glycerol–water mixtures up to 30% glycerol.