MECHANISM OF CYTOCHROME-P450 2D6-CATALYZED SPARTEINE METABOLISM IN HUMANS

Two different reaction mechanisms for the formation of the two human e namine-structured sparteine metabolites by cytochrome P450 2D6 have be en discussed in the literature. These mechanisms are either initial on e-electron oxidation of N1 of sparteine followed by deprotonation of t he aminium radical cation, resulting in the formation of different car bon radicals and oxygen rebound of the carbon radicals, or oxidation o f the carbon atoms adjacent to N1 by the enzyme, directly producing th e respective carbon radicals. With a spectrum of deuterium-labeled iso topomers of sparteine, stereoselectivity and kinetic isotope effects o f human sparteine metabolism were investigated by in vitro and in vivo experiments and were compared with chemical oxidation of 17-oxosparte ine. These experiments revealed that the major human sparteine metabol ite 2,3-didehydrosparteine is formed via highly stereoselective abstra ction of the 2 beta-hydrogen atom; the deuterium label was completely retained during metabolism when 2R-[H-2]sparteine was used as substrat e. Chemical oxidation of 17-oxosparteine by Ce4+ as a model for one-el ectron 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 fo und. An unequivocal discrimination between the two possible reaction m echanisms was not possible by simple interpretation of the magnitude o f the kinetic deuterium isotope effects, However, results of competiti ve and noncompetitive experiments revealed the presence of a nondissoc iative enzymatic mechanism for the formation of the two sparteine meta bolites, i.e., the sparteine molecule that is bound to the substrate b inding site of cylochrome P450 2D6 performs orientational changes with out dissociating from the activated enzyme/substrate complex before th e 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 o ccur over some distance, the possibility of a sequential electron-prot on transfer reaction during sparteine metabolism cannot be ruled out c ompletely as an alternative reaction mechanism for sparteine metabolis m.