PT  - JOURNAL ARTICLE
AU  - Hsia-lien Lin
AU  - Ute M. Kent
AU  - Paul F. Hollenberg
TI  - Mechanism-Based Inactivation of Cytochrome P450 3A4 by 17α-Ethynylestradiol: Evidence for Heme Destruction and Covalent Binding to Protein
AID  - 10.1124/jpet.301.1.160
DP  - 2002 Apr 01
TA  - Journal of Pharmacology and Experimental Therapeutics
PG  - 160--167
VI  - 301
IP  - 1
4099  - http://jpet.aspetjournals.org/content/301/1/160.short
4100  - http://jpet.aspetjournals.org/content/301/1/160.full
SO  - J Pharmacol Exp Ther2002 Apr 01; 301
AB  - 17α-Ethynylestradiol (EE), a major constituent of many oral contraceptives, inactivated the testosterone 6β-hydroxylation activity of purified P450 3A4 reconstituted with phospholipid and NADPH-cytochrome P450 reductase in a mechanism-based manner. The inactivation of P450 3A4 followed pseudo first order kinetics and was dependent on NADPH. The values for the KIand kinact were 18 μM and 0.04 min−1, respectively, and thet1/2 was 16 min. Incubation of 50 μM EE with P450 3A4 at 37°C for 30 min resulted in a 67% loss of testosterone 6β-hydroxylation activity accompanied by a 35% loss of the spectral absorbance of the native protein at 415 nm and a 70% loss of the spectrally detectable P450-CO complex. The inactivation of P450 3A4 by EE was irreversible. Testosterone, an alternate substrate, was able to protect P450 3A4 from EE-dependent inactivation. The partition ratio was ∼50. The stoichiometry of binding was approximately 1.3 nmol of an EE metabolite bound per nmol of P450 3A4 inactivated. SDS-polyacrylamide gel electrophoresis analysis demonstrated that [3H]EE was irreversibly bound to the P450 3A4 apoprotein. After extensive dialysis of the [3H]EE inactivated samples, high-pressure liquid chromatography (HPLC) analysis demonstrated that the inactivation resulting from EE metabolism led to the destruction of approximately half the heme with the concomitant generation of modified heme and EE-labeled heme fragments and produced covalently radiolabeled P450 3A4 apoprotein. Electrospray mass spectrometry demonstrated that the fraction corresponding to the major radiolabeled product of EE metabolism has a mass (M − H)− of 479 Da. HPLC and gas chromatography-mass spectometry analyses revealed that EE metabolism by P450 3A4 generated one major metabolite, 2-hydroxyethynylestradiol, and at least three additional metabolites. In conclusion, our results demonstrate that EE is an effective mechanism-based inactivator of P450 3A4 and that the mechanism of inactivation involves not only heme destruction, but also the irreversible modification of the apoprotein at the active site. The American Society for Pharmacology and Experimental Therapeutics