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.
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.
Molinié
Roland
R
Laboratory of Isotopic and Electrochemical Analysis of Metabolism (LAIEM), CNRS UMR6006, University of Nantes, 2 rue de la Houssinière, 44322 Nantes, France.
Kwiecień
Renata A
RA
Paneth
Piotr
P
Hatton
Wilfried
W
Lebreton
Jacques
J
Robins
Richard J
RJ
D003903:Deuterium / Q000378:metabolism; D006878:Hydroxides / Q000378:metabolism*; D008745:Methylation; D008956:Models, Chemical; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D009538:Nicotine / Q000378:metabolism*; D009587:Nitrogen Isotopes / Q000378:metabolism; D011759:Pyrrolidines / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology / Q000378:metabolism*
D006878:Hydroxides; C518264:N-methyl-2-phenylpyrrolidine; D009587:Nitrogen Isotopes; D011759:Pyrrolidines; D009538:Nicotine; C031356:hydroxide ion; D003903:Deuterium; D009251:NADPH-Ferrihemoprotein Reductase