title: Substrate probe for the mechanism of aromatic hydroxylation catalyzed by cytochrome P450.
abstract: The effect of branch pathways on the observed intramolecular isotope effect and deuterium retention associated with 6- and 7-hydroxylation of selectively monodeuterated (R)- and (S)-warfarin with cytochrome P450 (CYP) 2C9 and CYP1A2 were studied. cDNA-expressed CYP2C9 was incubated with enantiomerically pure (S)-7d1- and (S)-6d1-warfarin, and expressed CYP1A2 was incubated with enantiomerically pure (R)-7d1- and (R)-6d1-warfarin. A high degree of deuterium retention was observed in all metabolites, independent of the stereochemistry of the substrate or CYP isoform. No deuterium kinetic isotope effect was observed for the formation of 6-hydroxy- or 7-hydroxywarfarin in the case of the (S)-6d1-warfarin metabolism by CYP2C9, or for the formation of 6-hydroxy-, 7-hydroxy-, and 8-hydroxywarfarin in the case of the (R)-6d1-warfarin metabolism by CYP1A2. Deuterium isotope effects of 1.17 and 1.23 accompanied formation of 7-hydroxywarfarin from (S)-7d1-warfarin by CYP2C9 and from (R)-7d1-warfarin by CYP1A2, respectively. These observations are consistent with the addition-rearrangement pathway for aromatic hydroxylation, in which a triplet-like active oxygen species initially adds to the pi system, resulting in a tetrahedral intermediate. The intermediate subsequently rearranges to generate the phenol, the final product of the reaction.
authors.affiliation: Department of Medical Chemistry, School of Pharmacy, University of Washington, Seattle 98195, USA.
authors.forename: J F
authors.initials: JF
authors.lastname: Darbyshire
authors.forename: K R
authors.initials: KR
authors.lastname: Iyer
authors.forename: J
authors.initials: J
authors.lastname: Grogan
authors.forename: K R
authors.initials: KR
authors.lastname: Korzekwa
authors.forename: W F
authors.initials: WF
authors.lastname: Trager
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases; D019388:Cytochrome P-450 CYP1A2 / Q000737:chemistry; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D003903:Deuterium; D006900:Hydroxylation; D013237:Stereoisomerism; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000737:chemistry; D014859:Warfarin / Q000031:analogs & derivatives / Q000737:chemistry*
chemical_list: D014859:Warfarin; D003577:Cytochrome P-450 Enzyme System; D003903:Deuterium; C087837:7-hydroxywarfarin; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D019388:Cytochrome P-450 CYP1A2; D039181:Steroid 16-alpha-Hydroxylase; C008091:6-hydroxywarfarin