Fatty acid discrimination and omega-hydroxylation by cytochrome P450 4A1 and a cytochrome P4504A1/NADPH-P450 reductase fusion protein.
Fatty acid discrimination and omega-hydroxylation by cytochrome P450 4A1 and a cytochrome P4504A1/NADPH-P450 reductase fusion protein.
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DOI：
10.1016/0003-9861(95)90012-8
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发表时间：
1995-07
影响因子：
3.9
通讯作者：
M. Alterman;C. Chaurasia;P. Lu;J. Hardwick;R. Hanzlik
中科院分区：
文献类型：
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作者：
M. Alterman;C. Chaurasia;P. Lu;J. Hardwick;R. Hanzlik
某些细胞色素 P450 酶对脂肪酸的 ω-羟基化显示出一定程度的链长和区域特异性，鉴于这些底物的构象灵活性、同系物之间性质的强烈相似性以及缺乏引导和加强酶-底物相互作用的极性基团（羧基末端除外），这一点非常显着。为了研究 ω-羟基化这些特征的化学基础，我们设计并合成了一系列月桂酸类似物，并评估它们作为细胞色素 P4504A1 和细胞色素 P450 4A1/NADPH-P450 还原酶融合蛋白催化的 ω-羟基化的底物和抑制剂。在正链烷酸中，月桂酸被发现具有最适合融合蛋白的链长，就像它对于天然细胞色素 P450 4A1 的链长一样。对于这两种酶，链缩短导致营业额急剧下降，而链延长则导致营业额逐渐下降。融合蛋白ω-羟基化月桂酸甲酯和月桂醇的效率约为月桂酸的十分之一，但它不会羟基化月桂酰胺。 10-甲氧基癸酸经历 O-去甲基化（通过 ω-羟基化）。支化底物 11-甲基月桂酸在 ω 位有效且选择性地羟基化。相比之下，环丙基类似物11,12-甲醇月桂酸并未被检测到羟基化，尽管它在等摩尔浓度下诱导I型结合谱并抑制月桂酸ω-羟基化43%。 ω-(咪唑基)-癸酸诱导 II 型血红素结合谱，是月桂酸羟基化的特别有效的抑制剂。总的来说，这些数据表明细胞色素 P450 4A1 的活性位点具有一定长度（约 14 Å）的细长管状形状，在其入口处具有极性基团（包括但不限于羧基）的识别位点，在其末端具有（氧代）血红素基团的识别位点。
The ω-hydroxylation of fatty acids by certain cytochrome P450 enzymes shows a degree of chain-length and regiospecificity which is remarkable in view of the conformational flexibility of these substrates, the strong similarity in properties among homologs, and the lack of polar groups (other than the carboxy terminus) with which to guide and strengthen enzyme-substrate interactions. To investigate the chemical basis for these features of ω-hydroxylation we designed and synthesized a series of lauric acid analogs and evaluated them as substrates and inhibitors of ω-hydroxylation catalyzed by cytochrome P4504A1 and a cytochrome P450 4A1/NADPH-P450 reductase fusion protein. Among n-alkanoic acids, lauric acid was found to have the optimum chain length for the fusion protein, as it does for native cytochrome P450 4A1. With both enzymes, chain shortening caused a precipitous drop in turnover while chain lengthening caused a gradual drop in turnover. The fusion protein ω-hydroxylated methyl laurate and lauryl alcohol about 1 10 th as efficiently as lauric acid, but it did not hydroxylate lauramide. 10-Methoxydecanoic acid underwent O-demethylation (via ω-hydroxylation). The branched substrate 11-methyllauric acid was hydroxylated efficiently and selectively at the ω-position. In contrast, the cyclopropyl analog 11,12-methanolauric acid was not detectably hydroxylated, although it induced Type I binding spectrum and inhibited lauric acid ω-hydroxylation by 43% at equimolar concentrations. ω-(Imidazolyl)-decanoic acid induced a Type II heme-binding spectrum and was an especially potent inhibitor of lauric acid hydroxylation. Collectively these data suggest that the active site of cytochrome P450 4A1 has an elongated tubular shape of definite length (ca. 14 Å) with a recognition site for polar groups (including but not limited to carboxyl) at its entrance and the (oxo)heme group at its terminus.