| Chloroperoxidase and horseradish peroxidase were found to catalyze the N-demethylation of several secondary and tertiary N-methyl arylamines supported by a variety of oxidants. For the peroxidase-catalyzed demethylation of N,N-dimethylaniline, the stoichiometry of formaldehyde formation and N-methylaniline formation was 1:1. Neither enzyme catalyzed the demethylation of N,N-dimethylaniline-N-oxide, indicating that the reaction does not proceed via an N-oxide intermediate. The results of initial rate and inhibition studies of the chloroperoxidase-catalyzed N-demethylation of N,N-dimethylaniline supported by ethyl hydroperoxide were consistent with a ping-pong mechanism for the reaction involving the oxidized enzyme compound I. When the N-methyl groups of N,N-dimethylaniline were deuterated, isotope effects on the rate constant for the interaction of the hydroperoxide substrate with the peroxidase in the demethylation reaction ranged from 1.7 to 5.1 depending upon the identity of the hydroperoxide. These results are best explained by a mechanism in which hydrogen (deuterium) is transferred from N,N-dimethylaniline to the enzyme during catalysis and must subsequently be displaced by the hydroperoxide on the next turnover. Isotope effects on the maximal velocity of the demethylation reaction suggested that carbon-hydrogen bond cleavage is a slow step relative to other steps in the horseradish peroxidase-catalyzed reaction while in the chloroperoxidase-catalyzed reaction this step is not slow. Deuterium solvent isotope effects on the demethylation reaction indicated that the interaction of the hydroperoxide with the enzyme is a slow step relative to other steps in the chloroperoxidase-catalyzed reaction while it is not slow in the horseradish peroxidase-catalyzed reaction. An intramolecular isotope effect of 10 was observed on the horseradish peroxidase-catalyzed demethylation of N-methyl, N-trideuteromethylaniline. This large isotope effect is indicative of complete bond cleavage in the transition state and these results are interpreted in terms of a chemical mechanism for the demethylation reaction involving hydrogen abstraction from N,N-dimethylaniline by peroxidase compound I to form an enzyme-bound N,N-dimethylaniline radical. |
