Stresser D M, Kupfer D
Worcester Foundation for Biomedical Research, Shrewsbury, Massachusetts 01545, USA.
Biochemistry. 1997 Feb 25;36(8):2203-10. doi: 10.1021/bi962129k.
CYP3A4 is the major human cytochrome P-450 in a superfamily of heme-thiolate proteins that catalyze the oxidation of numerous lipophilic compounds. In this investigation, we report that CYP3A4 requires a phenolic function for ortho hydroxylation of estradiol and mono-O-demethylated methoxychlor and that CYP3A4 aromatic hydroxylation in general may be dependent on the presence of a free phenolic group. Indeed, when methoxyls were present instead of phenolic hydroxyls, CYP3A4 essentially failed to catalyze ortho hydroxylation. By contrast, of eight additional cDNA-expressed P-450s (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2D6, and 2E1) examined, only CYP1A2 and CYP2B6 could catalyze ortho hydroxylation of [o-3H]methoxychlor (7.2 and 14.6 pmol/90 min/pmol P-450, respectively), indicating that these isoforms do not require a phenolic hydroxyl for aromatic hydroxylation and that methoxyls do not sterically hinder catalysis by these CYPs. However, with [o-3H]mono-O-demethylated methoxychlor, containing a phenolic group, five isoforms (CYP1A2, 2B6, 2D6, 2E1, and 3A4) supported ortho hydroxylation. Of these, CYP3A4 exhibited by far the highest rate of hydroxylation at 87.8 pmol/90 min/pmol P-450. Further studies with [2-(3)H]estradiol 3-methyl ether and with [2-(3)H]estradiol revealed a similar and dramatic augmentation of CYP3A4-mediated C2 hydroxylase activity of approximately 75-fold by the presence of the phenolic group in the 3-position. The mechanism of augmentation by the phenolic hydroxyl does not appear to involve the acidic proton of estradiol, since CYP3A4-catalyzed estradiol 2-hydroxylation and testosterone 6-beta-hydroxylation were diminished to an equal extent when incubations were performed at increasing buffer pH values from 7 to 9. Both estradiol and its 3-methoxy derivative bound with similar affinity to cDNA-expressed, microsomal CYP3A4: spectral dissociation constants were 270 and 370 microM, respectively, and both compounds exhibited type I spectra. Thus, the disparities in aromatic hydroxylation rates between compounds containing phenolic hydroxyls and those with methoxyls cannot be explained by differences in their binding affinities. To explain the mode via which the phenolic hydroxyl facilitates ortho hydroxylation, a mechanism in which the phenolic moiety attacks the iron-oxo double bond of CYP3A4, resulting in oxygen transfer to the ortho position, is proposed. It is anticipated that these findings will assist in forecasting the CYP-mediated metabolic fate of phenolic compounds.
细胞色素P450 3A4（CYP3A4）是血红素硫醇盐蛋白超家族中的主要人类细胞色素P450，可催化多种亲脂性化合物的氧化反应。在本研究中，我们报告称CYP3A4催化雌二醇和单-O-去甲基甲氧滴滴涕的邻位羟基化反应需要酚羟基官能团，并且一般而言CYP3A4的芳香族羟基化反应可能依赖于游离酚羟基的存在。实际上，当存在甲氧基而非酚羟基时，CYP3A4基本无法催化邻位羟基化反应。相比之下，在检测的另外8种cDNA表达的P450（CYP1A1、1A2、2A6、2B6、2C8、2C9、2D6和2E1）中，只有CYP1A2和CYP2B6能够催化[邻位-3H]甲氧滴滴涕的邻位羟基化反应（分别为7.2和14.6 pmol/90分钟/pmol P450），这表明这些同工酶在芳香族羟基化反应中不需要酚羟基，并且甲氧基在空间上不会阻碍这些细胞色素P450的催化作用。然而，对于含有酚基的[邻位-3H]单-O-去甲基甲氧滴滴涕，有5种同工酶（CYP1A2、2B6、2D6、2E1和3A4）支持邻位羟基化反应。其中，CYP3A4的羟基化速率最高，为87.8 pmol/90分钟/pmol P450。对[2-(3)H]雌二醇3-甲醚和[2-(3)H]雌二醇的进一步研究表明，3位存在酚基时，CYP3A4介导的C2羟化酶活性有类似且显著的增强，约为75倍。酚羟基增强反应的机制似乎不涉及雌二醇的酸性质子，因为当在缓冲液pH值从7增加到9的条件下进行孵育时，CYP3A4催化的雌二醇2-羟基化反应和睾酮6-β-羟基化反应同等程度地减弱。雌二醇及其3-甲氧基衍生物与cDNA表达的微粒体CYP3A4具有相似的结合亲和力：光谱解离常数分别为270和370 μM，并且两种化合物均表现出I型光谱。因此，含有酚羟基的化合物与含有甲氧基的化合物在芳香族羟基化速率上的差异不能用它们结合亲和力的不同来解释。为了解释酚羟基促进邻位羟基化的方式，提出了一种机制，即酚基部分攻击CYP3A4的铁-氧双键，导致氧转移到邻位。预计这些发现将有助于预测酚类化合物的细胞色素P450介导的代谢命运。
