Inhibition of aromatase cytochrome P-450 by 10-oxirane and 10-thiirane substituted androgens. Implications for the structure of the active site.

The mechanism of inhibition of estrogen synthetase (P-450arom) by 19R- and 19S-isomers of 10-oxiranyl-and 10-thiiranyl-4-estrene-3,17-dione was investigated using human placental microsomes and purified enzyme preparations. The 19R-isomers were potent inhibitors and exhibited affinities 36-fold (10-oxirane) and 80-fold (10-thiirane) greater than the respective 19S-isomers. Kinetic experiments showed that inhibition by the 19R-isomers is competitive with respect to substrate; inhibition constants for the (19R)-10-oxirane (Ki = 10 nM) and the 19R-10-thiirane (Ki = 2 nM) indicate that each binds with greater affinity than the androgen substrates androstenedione and testosterone. Inhibition time courses and kinetic data were consistent with high affinity, reversible binding. Spectral titrations of microsomal preparations and purified P-450arom showed that binding of the 19R-isomers shifts the Soret maximum of the ferric enzyme to 411 nm (10-oxirane) or 425 nm (10-thiirane); addition of excess androstenedione reversed the spectral changes, producing the high spin form of the enzyme with a Soret peak at 393 nm. These spectral shifts suggest that the oxygen atom of the 10-oxirane and the sulfur atom of the 10-thiirane are bound to the heme iron in the inhibitor complexes. These results suggest that the high affinities of the inhibitors arise from their dual interaction with the androgen binding site and with the heme. Coordination of the C19 heteroatom to the heme indicates that C19 of androgen substrates may be positioned sufficiently close to the heme to allow direct attack by an iron-bound oxidant. Stereoselective binding of the 19R-isomers by P-450arom further suggests that the heme is likely to be positioned above C1 and C2 of the A ring.