Evidence for a 1-electron oxidation mechanism in N-dealkylation of N,N-dialkylanilines by cytochrome P450 2B1. Kinetic hydrogen isotope effects, linear free energy relationships, comparisons with horseradish peroxidase, and studies with oxygen surrogates.
Many enzymes catalyze N-dealkylations of alkylamines, including cytochrome P450 (P450) and peroxidase enzymes. Peroxidases, exemplified by horseradish peroxidase (HRP), are generally accepted to catalyze N-dealkylations via 1-electron transfer processes. Several lines of evidence also support a 1-electron mechanism for many P450 reactions, although this view has been questioned in light of reported trends for kinetic hydrogen isotope effects for N-demethylation with a series of 4-substituted N,N-dimethylanilines. No continuous trend for an increase of isotope effects with the electronic parameters of para-substitution was seen for the P450 2B1-catalyzed reactions in this study. The larger value seen with the 4-nitro derivative is consistent with a shift in mechanism due to either a reversible electron transfer step preceding deprotonation or to a hydrogen atom abstraction mechanism. With HRP, the trend is to lower isotope effects with para electron-withdrawing substituents, due to an apparent shift in rate-limiting steps. Biomimetic model high-valent porphyrins showed reduction rates with variously 4-substituted N,N-dialkylanilines that were consistent with a positively charged intermediate; such relationships were not seen for anisole O-demethylation with P450 2B1. In contrast to the case with the NADPH-supported P450 reactions, high deuterium isotope effects ( approximately 7) were seen in the N-dealkylations supported by the oxygen surrogate iodosylbenzene. With iodosylbenzene, colored aminium radicals were observed in the oxidations of aminopyrine, N,N-dimethyl-4-aminothioanisole, and 4-methoxy-N,N-dimethylaniline. With the latter compound, a substantial intermolecular deuterium isotope effect was observed for N-demethylation. In the N-dealkylation of N-ethyl,N-methylaniline by P450 2B1 (NADPH-supported), the ratio of N-demethylation to N-deethylation was 16. Although it is probably possible for P450s to catalyze amine N-dealkylations via hydrogen atom abstraction when such a course is electronically or sterically favored, we interpret the evidence to favor a 1-electron pathway with N,N-dialkylamines with P450 2B1 as well as HRP and several biomimetic models.
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
F P
FP
Guengerich
C H
CH
Yun
T L
TL
Macdonald
D000478:Alkylation; D000814:Aniline Compounds / Q000378:metabolism*; D000818:Animals; D019362:Cytochrome P-450 CYP2B1 / Q000378:metabolism*; D004583:Electrons; D006735:Horseradish Peroxidase / Q000378:metabolism*; D007700:Kinetics; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D013379:Substrate Specificity
D000814:Aniline Compounds; D006735:Horseradish Peroxidase; D019362:Cytochrome P-450 CYP2B1; D010634:Phenobarbital