Cytochrome P450-Catalyzed Degradation of Nicotine: Fundamental Parameters Determining Hydroxylation by Cytochrome P450 2A6 at the 5鈥?Carbon or the N-Methyl Carbon

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• 作者：Renata A. Kwiecie艅 ; Jean-Yves Le Questel ; Jacques Lebreton ; Marcel Delaforge ; Fran莽ois Andr茅 ; Emilie Pihan ; Ana茂s Roussel ; Ana茂s Fournial ; Piotr Paneth ; Richard J. Robins
• 刊名：The Journal of Physical Chemistry B
• 出版年：2012
• 出版时间：July 12, 2012
• 年：2012
• 卷：116
• 期：27
• 页码：7827-7840
• 全文大小：630K
• 年卷期：v.116,no.27(July 12, 2012)
• ISSN：1520-5207

文摘

The oxidation of (2鈥?i>S)-nicotine in the active site of human cytochrome P450 2A6 has been subjected to a detailed analysis by theoretical quantum mechanical/molecular mechanical (QM/MM) calculations linked with a theoretical and experimental study of the associated isotope effects. The study has focused on seeking an explanation as to why oxidation at the 5鈥?carbon position (A) is favored over oxidation at the methyl carbon (C_Me) position (B). It is deduced that the choice of hydrogen for abstraction is not determined by geometric features of the active site, but by the lower energy barrier associated with 5鈥?oxidation. N-Demethylation leading to N-hydroxymethylnornicotine requires ca. 6.5 kcal/mol more energy to transfer a hydrogen atom than is required for oxidation on the carbon 5鈥? Neither protonation of the pyrrolidine nitrogen (N1鈥? nor inclusion of a water molecule in the reaction process influences the balance between the two oxidation pathways. In both cases, the hydrogen transfer step is rate limiting. An analysis of the calculated kinetic isotope effects indicates that the presence of a ²H in either the C5鈥?or the C_Mepositions has a significant effect on the reaction kinetics. However, the experimental values of around 2.2鈥?.6 are considerably lower than those predicted by theoretical calculations (9.3 and 6.9 for C5鈥?or the C_Me positions, respectively, in the LS state of Cpd I), typical of the masking commonly found for CYP450 reactions. The fact that similar values are found for cotinine formation from both substrates, however, may indicate that the measured value is not that for H-abstraction but, rather, is a combined value for ²H influence on electronic redistribution between iminium states of the pyrrolidine ring. This is the first time that oxidation at the C5鈥?or the C_Me positions has been directly compared and that isotope effects have been obtained for this reaction in a human cytochrome P450 reaction.