Conley Alan, Mapes Samantha, Corbin C Jo, Greger Douglas, Graham Sandra
Department of Population Health and Reproduction, University of California School of Veterinary Medicine, Davis, California 95616, USA.
Mol Endocrinol. 2002 Jul;16(7):1456-68. doi: 10.1210/mend.16.7.0876.
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.
猪性腺形式的芳香化酶细胞色素P450（P450arom）对咪唑类药物依托咪酯的抑制作用比胎盘同工酶更为敏感。通过嵌合诱变和胎盘同工酶的点突变分析确定了造成这种功能差异的残基，并对在昆虫细胞中过表达后的天然和突变酶进行了动力学分析。通过替换预测的底物识别位点-1（SRS-1），胎盘同工酶对依托咪酯的敏感性显著增加，而通过同时替换SRS-1和预测的B螺旋，基本上重现了性腺同工酶的敏感性。胎盘同工酶第133位的单个异亮氨酸（I）突变为甲硫氨酸（M）（I(133)M）被证明是SRS-1中的关键残基。第133位残基位于B'-C环中，并且已证明在其他类固醇代谢P450中同样重要。单点突变（包括第110、114、120、128、137位残基及其组合等）以及整个B和C螺旋的突变对依托咪酯抑制敏感性均无显著影响。引入人P450arom中的相同突变（I(133)M）也显著增加了依托咪酯敏感性。异亮氨酸（Ile）第133位突变为丙氨酸（I(133)A）或酪氨酸（I(133)Y）会降低表观酶活性，但I(133)A突变体对依托咪酯抑制敏感，这表明是Ile(133)降低了依托咪酯结合，而不是Met(133)增加了酶的敏感性。I(133)M突变体和天然胎盘同工酶的雄烯二酮周转和亲和力相似。这些数据表明Ile(133)是P450arom的SRS-1中的一个接触残基，强调了许多类固醇羟化P450酶的SRS-1中存在功能保守性，并表明底物和抑制剂结合在不同程度上依赖于不同的接触点。
