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Vol. 301, Issue 1, 160-167, April 2002
-Ethynylestradiol: Evidence for Heme Destruction and Covalent
Binding to Protein
Department of Pharmacology, University of Michigan, Ann Arbor,
Michigan
17
-Ethynylestradiol (EE), a major constituent of many oral
contraceptives, inactivated the testosterone 6
-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 KI
and kinact were 18 µM and 0.04 min
1, respectively, and the
t1/2 was 16 min. Incubation of 50 µM EE with P450 3A4 at 37°C for 30 min resulted in a 67% loss of
testosterone 6-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.
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