Evaluation of the mechanism of aromatase cytochrome P450 - A site-directed mutagenesis study
Evaluation of the mechanism of aromatase cytochrome P450 - A site-directed mutagenesis study
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DOI：
10.1046/j.1432-1033.2001.01886.x
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发表时间：
2001-01-01
期刊：
影响因子：
--
通讯作者：
Chen, S
中科院分区：
文献类型：
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作者：
Kao, YC;Korzekwa, KR;Chen, S
芳香化酶（CYP 19）催化三个连续的羟基化反应，将C19雄激素转化为芳香族C18雌激素类固醇。在这项研究中，五个人芳香化酶突变体（E302 D，S478 A，S478 T，H480 K，和H480 Q）使用哺乳动物细胞表达系统制备。通过酶动力学分析、抑制谱研究和反应中间体测量对这些突变体进行了评价。三种甾体抑制剂[4-羟基雄烯二酮（4-OHA），7 α-（4 '-氨基）苯硫基-1，4-雄甾二烯-3，17-二酮（7 α-APTADD），和桥（2，19-亚甲基氧基）雄甾烯-3，17-二酮（MDL 101003）]和4种非甾体抑制剂[氨鲁米特（AG）、CGS 20267、ICI D1033和伏罗唑（R83842）]用于抑制特征研究。我们的芳香化酶的计算机模型表明，Glu 302是位于保守的I-螺旋区域，位于附近的C-19位置的类固醇底物。动力学参数的显著变化和突变体E302 D的MDL 101，003的Ki值增加7倍支持该模型。由于发现S478 A具有与野生型酶相似的动力学性质，并且比S478 T具有高得多的活性，因此认为Ser 478位于相当受限的环境中。与野生型酶相比，S478 T的7 α-APTADD的Ki值增加了10倍，表明Ser 478可能靠近底物的C-7位置。反应中间体分析显示，与野生型酶相比，S478 A和S478 T均产生显著更多的19-醇中间体。这些结果将支持Ser 478在第一和第二羟基化反应中起作用的假设。如H480 K具有比H480 Q显著更高的活性的事实所示，带正电荷的氨基酸优选在位置480处。发现4-OHA对H480 Q的Ki值是野生型酶的三倍。此外，突变体H480 K和H480 Q比野生型酶检测到显著更多的19-醇和19-al中间体。在His 480的两个突变的评估允许我们提出，这个残基可能参与芳构化反应（第三步）作为底物的C-3酮基的氢键供体。此外，当酶在Ser 478和His 480处突变时，产生新的产物。因此，这两个残基必须在催化中发挥重要作用，并且可能比以前预测的更接近底物结合位点。
Aromatase (CYP19) catalyzes three consecutive hydroxylation reactions converting C19 androgens to aromatic C18 estrogenic steroids. In this study, five human aromatase mutants (E302D, S478A, S478T, H480K, and H480Q) were prepared using a mammalian cell expression system. These mutants were evaluated by enzyme kinetic analysis, inhibitory profile studies, and reaction intermediate measurements. Three steroidal inhibitors [4-hydroxyandrostenedione (4-OHA), 7 alpha-(4'-amino)phenylthio-1,4-androstandiene-3,17-dione (7 alpha -APTADD), and bridge (2,19-methyleneoxy) androstene-3,17-dione (MDL 101003)], and four nonsteroidal inhibitors [aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole (R83842)] were used in the inhibitory profile studies. Our computer model of aromatase suggests that Glu302 is situated in the conserved I-helix region and located near the C-19 position of the steroid substrate. The model was supported by significant changes in kinetic parameters and a sevenfold increase in the K-i value of MDL 101,003 for the mutant E302D. As S478A was found to have kinetic properties similar to the wild-type enzyme and a much higher activity than S478T, Ser478 is thought to be situated in a rather restricted environment. There was a 10-fold increase in the K-i value of 7 alpha -APTADD for S478T over that for the wild-type enzyme, suggesting that Ser478 might be near the C-7 position of the substrate. The reaction intermediate analysis revealed that significantly more 19-ol intermediate was generated by both S478A and S478T than the wild-type enzyme. These results would support a hypothesis that Ser478 plays a role in the first and second hydroxylation reactions. A positive charged amino acid is preferred at position 480 as shown by the fact that H480K has a significantly higher activity than H480Q. The K-i value of 4-OHA for H480Q was found to be three times that of the wild-type enzyme. In addition, significantly more 19-ol and 19-al intermediates were detected for both mutants H480K and H480Q than for the wild-type enzyme. Evaluation of the two mutations at His480 allows us to propose that this residue may participate in the aromatization reaction (the third step) by acting as a hydrogen bond donor for the C-3 keto group of the substrate. Furthermore, new products were generated when the enzyme was mutated at Ser478 and His480. Thus, these two residues must play an important role in the catalysis and are likely closer to the substrate binding site than previously predicted.