Iwasaki M, Lindberg R L, Juvonen R O, Negishi M
Pharmacogenetics Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709.
Biochem J. 1993 Apr 15;291 ( Pt 2)(Pt 2):569-73. doi: 10.1042/bj2910569.
We have cloned a cDNA encoding mouse steroid 7 alpha-hydroxylase P450(7) alpha (cytochrome P-450(7) alpha) and expressed it in Saccharomyces cerevisiae. Mouse P450(7) alpha is 70% identical in its amino acid sequence with the mouse steroid 15 alpha-hydroxylase P450(15) alpha (2A4). The Leu at position 209 of P450(15) alpha is the most important residue to determine the steroid hydroxylase activity of the P450 [Lindberg and Negishi (1989) Nature (London) 339, 632-634]. The P450(7) alpha contains Asn at the position corresponding to the Leu-209 of P450(15) alpha, although both P450s hydroxylate testosterone. The CO-reduced P450(7) alpha complex is unstable, so that it is quickly converted into the inactive P420, whereas the P450(15) alpha is very stable. The P450(7) alpha, however, is stabilized either by addition of testosterone or by a mutation of Asn-209 to Leu. The mutant P450(7) alpha displays a 17-fold lower Vmax. value than the wild-type enzyme. Unexpectedly, it also has 3-fold lower Km and Kd values. Residue 209 in P450(7) alpha, therefore, appears to be located at a critical site of the haem-substrate-binding pocket. Corticosterone inhibits the testosterone 7 alpha-hydroxylase activity of the wild-type P450(7) alpha, whereas it does not inhibit the mutant P450(7) alpha. Conversely, the P450(15) alpha activity becomes inhibited by corticosterone upon the replacement of Leu-209 by Asn. In addition, this mutation increases the corticosterone 15 alpha-hydroxylase activity of P450(15) alpha at least 20-fold. Whereas the inhibition by corticosterone depends on the presence of Asn at position 209, deoxycorticosterone inhibits the activities of the P450s regardless of the type of residue at 209. The results indicate, therefore, that the identity of residue 209 determines the affinity as well as specificity of steroid binding to both P450(7) alpha and P450(15) alpha.
我们克隆了编码小鼠甾体7α-羟化酶P450(7)α（细胞色素P-450(7)α）的cDNA，并在酿酒酵母中进行了表达。小鼠P450(7)α的氨基酸序列与小鼠甾体15α-羟化酶P450(15)α（2A4）有70%的同源性。P450(15)α第209位的亮氨酸是决定该P450甾体羟化酶活性的最重要残基[林德伯格和根岸（1989年），《自然》（伦敦）339, 632 - 634]。尽管两种P450都能使睾酮羟化，但P450(7)α在与P450(15)α的Leu - 209相对应的位置含有天冬酰胺。一氧化碳还原的P450(7)α复合物不稳定，会迅速转化为无活性的P420，而P450(15)α则非常稳定。然而，通过添加睾酮或使Asn - 209突变为亮氨酸，P450(7)α可得到稳定。突变型P450(7)α的Vmax值比野生型酶低17倍。出乎意料的是，它的Km和Kd值也低3倍。因此，P450(7)α中的第209位残基似乎位于血红素-底物结合口袋的关键位点。皮质酮抑制野生型P450(7)α的睾酮7α-羟化酶活性，而不抑制突变型P450(7)α。相反，当Leu - 209被Asn取代时，皮质酮会抑制P450(15)α的活性。此外，这种突变使P450(15)α的皮质酮15α-羟化酶活性至少提高20倍。虽然皮质酮的抑制作用取决于第209位天冬酰胺的存在，但脱氧皮质酮无论第209位残基的类型如何都会抑制这两种P450的活性。因此，结果表明第209位残基的特性决定了甾体与P450(7)α和P450(15)α结合的亲和力和特异性。
