OXIDATION OF DIMETHYLANILINE BY HORSERADISH-PEROXIDASE AND ELECTROGENERATED PEROXIDE .1. FREE ENZYME STUDIES

The N-demethylation of N,N-dimethylaniline (DMA) with free horseradish peroxidase (HRP) in solution was studied using electrogenerated hydrogen peroxide. The Michaelis-Menten constants with respect to varying DMA concentrations, determined from rates of N-demethylation using added hydrogen peroxide (peroxide-saturated enzyme conditions), were K(m) = 0.68 mM and V(max) = 6500 mol CH2O/min/mol HRP. Apparent Michaelis-Menten parameters, determined from rates at low concentrations of peroxide (approximately 5 muM) generated in situ by constant current oxygen reduction at a glassy carbon cathode, were K(m) = 0.19 and V(max) = 2 000; these values were consistent with calculations based on the peroxide-saturated results and a simple two-substrate Ping Pong mechanism. The optimum pH and temperature were determined to be 5.5 and 28-degrees-C, respectively.