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Radical cation intermediates in N-dealkylation reactions
F P Guengerich1, O Okazaki, Y Seto
1Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Summary
Cytochrome P450 enzymes catalyze N-dealkylation via sequential electron transfer (SET) and base catalysis, distinct from peroxidases. This mechanism explains isotope effects and lack of H218O incorporation in P450-mediated reactions.
Area of Science:
- Biochemistry
- Enzymology
- Organic Chemistry
Background:
- Cytochrome P450 enzymes (P450s) catalyze N-dealkylation through various proposed mechanisms, including C- and N-hydroxylation and sequential electron transfer (SET).
- Peroxidases strongly favor SET, with detectable free radicals, and exhibit high kinetic deuterium isotope effects in N-dealkylation.
- Key mechanistic distinctions between P450s and peroxidases include lack of H218O incorporation into products by P450s and differing kinetic deuterium isotope effects.
Purpose of the Study:
- To investigate the mechanistic possibilities of P450-catalyzed N-dealkylation, focusing on the role of SET and base catalysis.
- To differentiate the N-dealkylation mechanisms of P450s from those of peroxidases.
- To elucidate the specific catalytic entity responsible for deprotonation in P450-mediated reactions.
Main Methods:
- Analysis of kinetic deuterium isotope effects for N-dealkylation reactions catalyzed by P450s and peroxidases.
- Investigation of products from dihydropyridine oxidations and mechanism-based inhibition studies.
- Application of Hammett and Marcus analyses to rates of N-demethylation of para-substituted N,N-dimethylanilines.
- Utilized biomimetic metalloporphyrin models to mimic P450 active sites.
Main Results:
- Evidence supports SET in P450 amine oxidations, but high kinetic deuterium isotope effects, characteristic of peroxidases, were absent.
- P450s appear to catalyze the deprotonation of aminium radicals, with low isotope effects observed for both P450s and metalloporphyrin models.
- The deprotonation is attributed to the (FeO)2+ entity, suggesting it acts as a strong base, unlike the shielded FeO moiety in peroxidases.
- Both enzyme classes catalyzed N-demethylation, but only peroxidases generated stable aminium radicals.
Conclusions:
- P450-catalyzed N-dealkylation proceeds via SET followed by base catalysis, likely involving the (FeO)2+ entity, to deprotonate the aminium radical.
- This mechanism explains the observed low kinetic isotope effects and lack of H218O incorporation in P450 reactions.
- A common aminium radical intermediate is proposed for both N-oxygenation and N-dealkylation pathways in N,N-dialkylaniline oxidation.