Enthalpy change and mechanism of oxidation of o-phenylenediamine by hydrogen peroxide catalyzed by horseradish peroxidase

Abstract

The hydrogen peroxide-oxidation of o-phenylenediamine (OPD) catalyzed by horseradish peroxidase (HRP) at 37 °C in 50 mM phosphate buffer (pH 7.0) was studied by calorimetry. The apparent molar reaction enthalpy with respect to OPD and hydrogen peroxide were −447 ± 8 kJ mol −1 and −298 ± 9 kJ mol −1, respectively. Oxidation of OPD by H 2O 2 catalyzed by HRP (1.25 nM) at pH 7.0 and 37 °C follows a ping–pong mechanism. The maximum rate V max (0.91 ± 0.05 μM s −1), Michaelis constant for OPD K m,S (51 ± 3 μM), Michaelis constant for hydrogen peroxide K m, H 2 O 2 (136 ± 8 μM), the catalytic constant k cat (364 ± 18 s −1) and the second-order rate constants k +1 = (2.7 ± 0.3) × 10 6 M −1 s −1 and k +5 = (7.1 ± 0.8) × 10 6 M −1 s −1 were obtained by the initial rate method.