Mechanism-based inactivation of P450 2A6 by furanocoumarins
Biochemistry. 1998 Jul 14;37(28):10047-61. doi: 10.1021/bi980003c.
L L Koenigs 1 , W F Trager
1 Department of Medicinal Chemistry, University of Washington, Seattle 98195, USA.
DOI: 10.1021/bi980003c
Several furanocoumarins were tested for their ability to inhibit human P450 2A6 activity. The metabolites and conjugates formed from these furanocoumarins after incubation with reconstituted purified P450 2A6 in the absence and presence of exogenous nucleophiles were characterized by UV and LC/ESI-MS/MS analysis. The results suggest initial oxidation to form a furanoepoxide followed by hydrolytic attack, or attack of exogenous nucleophiles, to form dihydrofuranocoumarin products. Initial epoxidation is confirmed by the finding that a single 18O atom is incorporated into the 8-methoxypsoralen (8-MOP) and psoralen (P) dihydrodiol metabolites when the incubations are performed in the presence of H218O. In contrast, 19% of the dihydrodiol formed from 5-methoxypsoralen (5-MOP) involves incorporation of two 18O atoms, implicating a gamma-ketoenal intermediate in the formation of this metabolite. Thus, the structure of the reactive intermediate(s) formed is dictated by the intrinsic electronic properties of the parent compound. After exposure to [14C]-8-MOP and [14C]-5-MOP, SDS-PAGE and HPLC experiments, followed by radiometric detection, indicated that both P450 2A6 and P450 reductase were covalently modified in the purified system. In contrast, only P450 2A6 was covalently modified in a lymphablastoid cell line (GENTEST). With the purified system, partition ratios were higher (1.5-3.9X), and the ability to scavenge reactive intermediates with exogenous nucleophiles was greater. These results suggest that relative to the cell system, more reactive intermediates can escape, rather than bind to, the active site of purified reconstituted P450 2A6.
Aryl Hydrocarbon Hydroxylases*
Carbon Radioisotopes
Coumarins / metabolism
Coumarins / pharmacology*
Cytochrome P-450 CYP2A6
Cytochrome P-450 Enzyme Inhibitors*
Cytochrome P-450 Enzyme System / metabolism
Deuterium Oxide / metabolism
Enzyme Activation / drug effects
Methoxsalen / analogs & derivatives
Methoxsalen / metabolism
Methoxsalen / pharmacology
Microsomes, Liver / enzymology
Mixed Function Oxygenases / antagonists & inhibitors*
Mixed Function Oxygenases / metabolism
Cytochrome P-450 Enzyme Inhibitors
Cytochrome P-450 Enzyme System
Mixed Function Oxygenases
Aryl Hydrocarbon Hydroxylases
Deuterium Oxide
GM32165/GM/NIGMS NIH HHS/United States