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

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 H2O2 catalyzed by HRP (1.25 nM) at pH 7.0 and 37 °C follows a ping–pong mechanism. The maximum rate Vmax (0.91 ± 0.05 μM s−1), Michaelis constant for OPD Km,S (51 ± 3 μM), Michaelis constant for hydrogen peroxide Km,H2O2Km,H2O2 (136 ± 8 μM), the catalytic constant kcat (364 ± 18 s−1) and the second-order rate constants k+1 = (2.7 ± 0.3) × 106 M−1 s−1 and k+5 = (7.1 ± 0.8) × 106 M−1 s−1 were obtained by the initial rate method.