Structural determinants of aromatase cytochrome p450 inhibition in substrate recognition site-1.

Abstract

The porcine gonadal form of aromatase cytochrome P450 (P450arom) exhibits higher sensitivity to inhibition by the imidazole, etomidate, than the placental isozyme. The residue(s) responsible for this functional difference was mapped using chimeragenesis and point mutation analysis of the placental isozyme, and the kinetic analysis was conducted on native and mutant enzymes after overexpression in insect cells. The etomidate sensitivity of the placental isozyme was markedly increased by substitution of the predicted substrate recognition site-1 (SRS-1) and essentially reproduced that of the gonadal isozyme by substitution of SRS-1 and the predicted B helix. A single isoleucine (I) to methionine (M) substitution at position 133 of the placental isozyme (I(133)M) was proven to be the critical residue within SRS-1. Residue 133 is located in the B'-C loop and has been shown to be equally important in other steroid-metabolizing P450s. Single point mutations (including residues 110, 114, 120, 128, 137, and combinations thereof among others) and mutation of the entire B and C helixes were without marked effect on etomidate inhibitory sensitivity. The same mutation (I(133)M) introduced into human P450arom also markedly increased etomidate sensitivity. Mutation of Ile(133) to either alanine (I(133)A) or tyrosine (I(133)Y) decreased apparent enzyme activity, but the I(133)A mutant was sensitive to etomidate inhibition, suggesting that it is Ile(133) that decreases etomidate binding rather than Met(133) increasing enzyme sensitivity. Androstenedione turnover and affinity were similar for the I(133)M mutant and the native placental isozyme. These data suggest that Ile(133) is a contact residue in SRS-1 of P450arom, emphasize the functional conservation that exists in SRS-1 of a number of steroid-hydroxylating P450 enzymes, and suggest that substrate and inhibitor binding are dependent on different contact points to varying degrees.