Steady state kinetics of chloroperoxidase-catalyzed N-demethylation reactions.

Abstract

The kinetic mechanism of the chloroperoxidase-catalyzed N-demethylation of N,N-dimethylaniline supported by ethyl hydroperoxide was determined from initial rate and inhibition studies. When the concentration of N,N-dimethylaniline was varied in the presence of several different fixed concentrations of ethyl hydroperoxide, a double reciprocal plot of the data gave a series of parallel lines. Parallel lines were also observed when the data were plotted as a function of the concentration of ethyl hydroperoxide. A linear double reciprocal plot was obtained when the concentrations of both substrates were varied in a constant ratio. Competitive substrate inhibition was observed for N,N-dimethylaniline with a KI of 4.78 mM. Competitive substrate inhibition was also observed for ethyl hydroperoxide but the secondary replot of the slopes of the double reciprocal lines versus the concentrations of ethyl hydroperoxide intercepted the origin, indicating that no abortive binary complex between ethyl hydroperoxide and the enzyme was formed. These results suggested that the inhibition of the demethylation reaction by ethyl hydroperoxide was due to the reaction of ethyl hydroperoxide with chloroperoxidase compound I to evolve oxygen. N,N-Dimethylaniline inhibited the chloroperoxidase-catalyzed evolution of oxygen from ethyl hydroperoxide with a KI (0.111 mM) essentially identical with its KM for demethylation (0.122 mM). The inhibition of the demethylation reaction by 2,5-dimethylfuran was competitive with respect to N,N-dimethylaniline and uncompetitive with respect to ethyl hydroperoxide, consistent with dead-end inhibition in a ping-pong system. The results of the initial rate and inhibition studies are consistent with a Ping Pong Bi Bi mechanism as the minimal kinetic model for chloroperoxidase-catalyzed N-demethylation reactions. In this model, ethyl hydroperoxide reacts with chloroperoxidase to form the oxidized enzyme intermediate compound I with the concomitant release of ethanol. N,N-Dimethylaniline then binds to compound I and is oxidized, resulting in the formation of N-methylaniline and formaldehyde and the regeneration of the native peroxidase.