title: Mechanism of cytochrome P450 2D6-catalyzed sparteine metabolism in humans.
abstract: Two different reaction mechanisms for the formation of the two human enamine-structured sparteine metabolites by cytochrome P450 2D6 have been discussed in the literature. These mechanisms are either initial one-electron oxidation of N1 of sparteine followed by deprotonation of the aminium radical cation, resulting in the formation of different carbon radicals and oxygen rebound of the carbon radicals, or oxidation of the carbon atoms adjacent to N1 by the enzyme, directly producing the respective carbon radicals. With a spectrum of deuterium-labeled isotopomers of sparteine, stereoselectivity and kinetic isotope effects of human sparteine metabolism were investigated by in vitro and in vivo experiments and were compared with chemical oxidation of 17-oxosparteine. These experiments revealed that the major human sparteine metabolite 2,3-didehydrosparteine is formed via highly stereoselective abstraction of the 2 beta-hydrogen atom; the deuterium label was completely retained during metabolism when 2R-[2H]sparteine was used as substrate. Chemical oxidation of 17-oxosparteine by Ce4+, as a model for one-electron oxidation of N1 of a sparteine-like structure, resulted in the sole formation of the 5,6-unsaturated enamine, and no 2,3-unsaturated enamine, structurally equivalent to the human major metabolite, was found. An unequivocal discrimination between the two possible reaction mechanisms was not possible by simple interpretation of the magnitude of the kinetic deuterium isotope effects. However, results of competitive and noncompetitive experiments revealed the presence of a nondissociative enzymatic mechanism for the formation of the two sparteine metabolites, i.e., the sparteine molecule that is bound to the substrate binding site of cytochrome P450 2D6 performs orientational changes without dissociating from the activated enzyme/substrate complex before the product-determining first irreversible reaction step. These results agree with the hypothesis that sparteine metabolism proceeds by direct carbon oxidation. Because electron transfer from amines to P450 may occur over some distance, the possibility of a sequential electron-proton transfer reaction during sparteine metabolism cannot be ruled out completely as an alternative reaction mechanism for sparteine metabolism.
authors.affiliation: Dr. Margarete Fischer-Bosch-Institut für Klinische Pharmakologie, Stuttgart, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Ebner
authors.forename: C O
authors.initials: CO
authors.lastname: Meese
authors.forename: M
authors.initials: M
authors.lastname: Eichelbaum
mesh_terms: D001667:Binding, Competitive; D002384:Catalysis; D019389:Cytochrome P-450 CYP2D6; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003903:Deuterium; D006801:Humans; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D010084:Oxidation-Reduction; D013034:Sparteine / Q000031:analogs & derivatives / Q000378:metabolism* / Q000652:urine; D013237:Stereoisomerism
chemical_list: D013034:Sparteine; D003577:Cytochrome P-450 Enzyme System; D003903:Deuterium; D006899:Mixed Function Oxygenases; D019389:Cytochrome P-450 CYP2D6; C035707:17-oxosparteine