Peroxidase-catalyzed N-demethylation reactions. Substrate deuterium isotope effects.
Deuterium isotope effects on the kinetic parameters for the hydroperoxide-supported N-demethylation of N,N-dimethylaniline catalyzed by chloroperoxidase and horseradish peroxidase were determined using N,N-di-(trideuteromethyl)aniline. The isotope effect on the Vmax for the chloroperoxidase-catalyzed demethylation reaction supported by ethyl hydroperoxide was 1.42 +/- 0.31. The isotope effects on the Vmax for the horseradish peroxidase-catalyzed reaction supported by ethyl hydroperoxide and hydrogen peroxide were 1.99 +/- 0.39 and 4.09 +/- 0.27, respectively. Isotope effects ranging from 1.76 to 5.10 were observed on the Vmax/Km for the hydroperoxide substrate (i.e. the second order rate constant for the reaction of the hydroperoxide with the peroxidase to form compound I) in both enzyme systems when the N-methyl groups of N,N-dimethylaniline were deuterated. These results are not predicted by the simple ping-pong kinetic model for peroxidase-catalyzed N-demethylation reactions. The data are most simply explained by a mechanism involving the transfer of deuterium (or hydrogen) from N,N-dimethylaniline to the enzyme during catalysis. The deuterium must subsequently be displaced from the enzyme by the hydroperoxide, causing the observed isotope effects.
D000814:Aniline Compounds; D002711:Chloride Peroxidase / Q000378:metabolism*; D003903:Deuterium; D006735:Horseradish Peroxidase / Q000378:metabolism*; D007700:Kinetics; D003904:Mitosporic Fungi / Q000201:enzymology; D010544:Peroxidases / Q000378:metabolism*; D013379:Substrate Specificity
D000814:Aniline Compounds; C015157:N,N-dimethylaniline; D003903:Deuterium; D006735:Horseradish Peroxidase; D010544:Peroxidases; D002711:Chloride Peroxidase