title: Mechanism-based inactivation of human liver cytochrome P450 2A6 by 8-methoxypsoralen.
abstract: The P450 2A6 catalyzed 7-hydroxylation of coumarin proceeded with a mean Km of 0.40 (+/-0.13) microM and Vmax of 6.34 nmol/nmol P450/min (36-fold variation) in microsomal preparations from a panel of 12 human livers. Substrate depletion was avoided during the kinetic determinations. 8-Methoxypsoralen (8-MOP) is a potent mechanism-based inactivator of human liver P450 2A6 and reconstituted purified recombinant P450 2A6 based on the following evidence: 1) 8-MOP causes time, concentration, and NADPH-dependent loss of P450 2A6 activity that is not reversed by potassium ferricyanide or extensive dialysis, 2) loss of P450 2A6 activity is associated with a loss of spectrally observable P450, 3) addition of nucleophiles or reactive oxygen scavengers to the incubations does not prevent inactivation of P450 2A6, and 4) 8-MOP-dependent P450 2A6 inactivation is inhibited (concentration dependent) by the addition of a competitive inhibitor (pilocarpine). Inactivation is selective for P450 2A6 at low concentrations of 8-MOP (2.5 microM) after short incubation time periods (3 min) and was characterized by a KI of 0.8 and 1.9 microM in a reconstituted and microsomal system, respectively, and a kinact of 1 min-1 and 2 min-1 in a reconstituted and microsomal system, respectively. A substrate depletion partition ratio of 21 was calculated for the inactivation of recombinant P450 2A6. Potency and selectivity suggest that 8-MOP could be a useful tool in vitro for evaluating P450 2A6 activity in various enzyme preparations.
authors.affiliation: Department of Medicinal Chemistry, University of Washington, Seattle, WA 98195, USA.
authors.forename: L L
authors.initials: LL
authors.lastname: Koenigs
authors.forename: R M
authors.initials: RM
authors.lastname: Peter
authors.forename: S J
authors.initials: SJ
authors.lastname: Thompson
authors.forename: A E
authors.initials: AE
authors.lastname: Rettie
authors.forename: W F
authors.initials: WF
authors.lastname: Trager
mesh_terms: D000293:Adolescent; D000328:Adult; D001189:Aryl Hydrocarbon Hydroxylases; D002648:Child; D003374:Coumarins / Q000378:metabolism; D019388:Cytochrome P-450 CYP1A2; D065609:Cytochrome P-450 CYP1A2 Inhibitors; D065637:Cytochrome P-450 CYP2A6; D065731:Cytochrome P-450 CYP2C19; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D065691:Cytochrome P-450 CYP2E1 Inhibitors; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D015786:Cytochromes b5 / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D006801:Humans; D007700:Kinetics; D008297:Male; D008730:Methoxsalen / Q000494:pharmacology*; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D008875:Middle Aged; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors; D013997:Time Factors
chemical_list: D003374:Coumarins; D065609:Cytochrome P-450 CYP1A2 Inhibitors; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D065691:Cytochrome P-450 CYP2E1 Inhibitors; D065607:Cytochrome P-450 Enzyme Inhibitors; D011994:Recombinant Proteins; D009249:NADP; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; C030123:coumarin; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C504573:CYP1A2 protein, human; C045793:CYP2C19 protein, human; C104464:CYP3A protein, human; D019388:Cytochrome P-450 CYP1A2; D065637:Cytochrome P-450 CYP2A6; D065731:Cytochrome P-450 CYP2C19; D051544:Cytochrome P-450 CYP3A; D039181:Steroid 16-alpha-Hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase; D008730:Methoxsalen