Mechanism-Based Inactivation of Human Liver Cytochrome P450 2A6 by 8-Methoxypsoralen

• Published in: Drug metabolism and disposition 1997-12, Vol.25 (12), p.1407-1415
• Main Authors: Koenigs, Luke L., Peter, Raimund M., Thompson, Stella J., Rettie, Allan E., Trager, William F.
• Format: Article
• Language: English
• Subjects: Adolescent; Adult; Analytical, structural and metabolic biochemistry; Aryl Hydrocarbon Hydroxylases; Biological and medical sciences; Child; Coumarins - metabolism; Cytochrome P-450 CYP1A2; Cytochrome P-450 CYP1A2 Inhibitors; Cytochrome P-450 CYP2A6; Cytochrome P-450 CYP2C19; Cytochrome P-450 CYP2D6 Inhibitors; Cytochrome P-450 CYP2E1 Inhibitors; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Cytochromes b5 - metabolism; Enzyme Activation - drug effects; Enzymes and enzyme inhibitors; Female; Fundamental and applied biological sciences. Psychology; Humans; Kinetics; Male; Medical sciences; Methoxsalen - pharmacology; Microsomes, Liver - drug effects; Microsomes, Liver - enzymology; Microsomes, Liver - metabolism; Middle Aged; Mixed Function Oxygenases - antagonists & inhibitors; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; NADP - metabolism; NADPH-Ferrihemoprotein Reductase - metabolism; Oxidoreductases; Pharmacology. Drug treatments; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Skin, nail, hair, dermoskeleton; Spectrophotometry; Steroid 16-alpha-Hydroxylase; Steroid Hydroxylases - antagonists & inhibitors; Time Factors
• ISSN: 0090-9556; 1521-009X
• DOI: 10.1124/dmd.25.12.1407

The P450 2A6 catalyzed 7-hydroxylation of coumarin proceeded with a mean Km of 0.40 (±0.13) μM 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 μM) after short incubation time periods (3 min) and was characterized by a KI of 0.8 and 1.9 μM 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.