pH kinetic studies of the N-demethylation of N,N-dimethylaniline catalyzed by chloroperoxidase.

The effect of pH on the kinetic parameters for the chloroperoxidase-catalyzed N-demethylation of N,N-dimethylaniline supported by ethyl hydroperoxide was investigated from pH 3.0 to 7.0. Chloroperoxidase was found to be stable throughout the pH range studied. Initial rate conditions were determined throughout the pH range. The Vmax for the demethylation reaction exhibited a pH optimum at approximately 4.5. The Km for N,N-dimethylaniline increased with decreasing pH, while the Km for ethyl hydroperoxide varied in a manner paralleling Vmax. Comparison of the Vmax/Km values for N,N-dimethylaniline and ethyl hydroperoxide indicated that the interaction of N,N-dimethylaniline with chloroperoxidase compound I was rate-limiting below pH 4.5, while compound I formation was rate-limiting above pH 4.5. The log of the Vmax/Km for ethyl hydroperoxide was independent of pH, indicating that chloroperoxidase compound I formation is not affected by ionizations in this pH range. The plot of the log of the Vmax/Km for N,N-dimethylaniline versus pH indicated an ionization on compound I with a pK of approximately 6.8. The plot of the log of the Vmax versus pH indicated an ionization on the compound I-N,N-dimethylaniline complex, with a pK of approximately 3.1. The results show that chloroperoxidase can demethylate both the protonated and neutral forms of N,N-dimethylaniline (pK approximately 5.0), suggesting that hydrophobic binding of the arylamine substrate is more important in catalysis than ionic bonding of the amine moiety. For optimal catalysis, a residue in the chloroperoxidase compound I-N,N-dimethylaniline complex with a pK of approximately 3.1 must be deprotonated, while a residue in compound I with a pK of approximately 6.8 must be protonated.