Abstract
The P450 2A6 catalyzed 7-hydroxylation of coumarin proceeded with a mean Km of 0.40 (±0.13) μM andVmax 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 akinact 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.
Footnotes
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Send reprint requests to: Dr. William F. Trager, Department of Medicinal Chemistry, Box 357610, University of Washington, Seattle, WA 98195. E-mail: trager{at}u.washington.edu.
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This research was supported by National Institutes of Health Grant GM32165 (to W.F.T.).
- Abbreviations used are::
- P450
- cytochrome P450
- HPLC
- high pressure liquid chromatography
- ACN
- acetonitrile
- DLPC
- L-d-phosphatidylcholine, dilauryl
- 8-MOP
- 8-methoxypsoralen
- NADPH-GS
- NADPH-generating system
- Received June 25, 1997.
- Accepted September 4, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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