Investigation of the mechanism of nicotine demethylation in Nicotiana through 2H and 15N heavy isotope effects: Implication of cytochrome P450 oxidase and hydroxyl ion transfer
| Autor: | Richard J. Robins, Wilfried Hatton, Renata A. Kwiecień, Roland Molinié, Jacques Lebreton, Piotr Paneth |
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| Rok vydání: | 2007 |
| Předmět: |
Nicotine
Pyrrolidines Biophysics Photochemistry Kinetic energy Methylation Biochemistry chemistry.chemical_compound Computational chemistry Tobacco Kinetic isotope effect Hydroxides Molecular Biology NADPH-Ferrihemoprotein Reductase Nicotiana Demethylation Nitrogen Isotopes biology Iminium Cytochrome P450 Deuterium biology.organism_classification Models Chemical chemistry biology.protein Methyl group SAM1 |
| Zdroj: | Archives of Biochemistry and Biophysics. 458:175-183 |
| ISSN: | 0003-9861 |
| DOI: | 10.1016/j.abb.2006.12.013 |
| Popis: | Heavy-atom isotope effects for the N-demethylation of nicotine have been determined in vivo in static-phase biosynthetically incompetent plant cell cultures of Nicotiana species. A (2)H kinetic isotope effect of 0.587 and a (15)N kinetic isotope effect of 1.0028 were obtained. An identical (15)N kinetic isotope effect of 1.0032 was obtained for the nicotine analogue, N-methyl-2-phenylpyrrolidine. The magnitude of the (15)N heavy-atom isotope effect indicates that the fission of the CN bond is not rate limiting for demethylation. The theoretical calculation of heavy-atom isotope effects for a model of the reaction pathway based on cytochrome P450 best fits the measured kinetic isotope effect to the addition of hydroxyl ion to iminium to form N-hydroxymethyl, for which the computed (2)H- and (15)N kinetic isotope effects are 0.689 and 1.0081, respectively. This large inverse (2)H kinetic isotope effect is not compatible with the initial abstraction of the H from the methyl group playing a significant kinetic role in the overall kinetic limitation of the reaction pathway, since computed values for this step (4.54 and 0.9995, respectively) are inconsistent with the experimental data. |
| Databáze: | OpenAIRE |
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