Elucidation of distinct ligand binding sites for cytochrome P450 3A4
Elucidation of distinct ligand binding sites for cytochrome P450 3A4
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DOI：
10.1021/bi992765t
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发表时间：
2000-05-23
期刊：
影响因子：
2.9
通讯作者：
Guengerich, FP
中科院分区：
文献类型：
--
作者：
Hosea, NA;Miller, GP;Guengerich, FP
细胞色素 P450 (P450) 3A4 是最丰富的人类 P450 酶，对底物具有广泛的选择性。该酶可以表现出显着的催化区域选择性和不寻常的同向和异向协同模式，为此提出了几种模型。光谱滴定研究表明药物茚地那韦（M-r 614）有一个结合位点，茚地那韦是一种已知的底物和抑制剂。几种 C 端胺化肽，包括模型吗啡肽 (YPFP-NH2)，与表明 Fe-NH2 键合的光谱变化结合。 YPFP-NH2 N 端胺的结合以及 C 端修饰对结合的影响表明整个分子 (M-r 521) 适合 P450 3A4。 YPFP-NH2 不会被 P450 3A4 氧化，但会阻断底物睾酮和咪达唑仑的结合，K-i 值与 YPFP-NH2 的光谱结合常数 (K-s) 相似。 YPFP-NH2 抑制几种典型 P450 底物的氧化，其 K-i 值比结合 YPFP-NH2 的 K-s 及其抑制底物结合的 K-i 大 10 倍。 YPFP-NH2 不会改变这些氧化协同性的 n 值，YPFP-NH2 在两个不同位置（1'- 和 4-）抑制咪达唑仑的氧化，K-i 值相差 20 倍。阻断与三价铁 P450 3A4 结合和几种底物氧化的 K-i 值的差异可能归因于 YPFP-NH2 与亚铁 P450 3A4 的结合比与三价铁形式的结合更弱。亚铁蛋白可以被认为是酶在结合和催化方面的一种独特形式，因为许多底物（但不是 YPFP-NH2）有助于将三价铁酶还原为亚铁酶。我们对这些肽的结果是在几个提出的模型的背景下考虑的。基于这些肽研究的 P450 3A4 模型包含至少两个、可能三个不同的配体位点，其中睾酮和 α-萘黄酮占据不同的位点。咪达唑仑似乎能够以两种模式与 P450 3A4 结合，一种对应于睾酮结合模式，另一种假定反映在第三个位点的结合，与睾酮和 α-萘黄酮都不同。茚地那韦和 YPFP-NH2 的研究还认为，P450 3A4 中应该存在空间来结合“睾酮”位点中的多个较小配体，尽管不存在这种结合的直接证据。尽管这项对肽的研究提供了多个配体结合位点存在的证据，但结果不能用于表明它们的并置，这可能会随着催化循环而变化。
Cytochrome P450 (P450) 3A4 is the most abundant human P450 enzyme and has broad selectivity for substrates. The enzyme can show marked catalytic regioselectivity and unusual patterns of homotropic and heterotropic cooperativity, for which several models have been proposed. Spectral titration studies indicated one binding site for the drug indinavir (M-r 614), a known substrate and inhibitor. Several C-terminal aminated peptides, including the model morphiceptin (YPFP-NH2), bind with spectral changes indicative of Fe-NH2 bonding. The binding of the YPFP-NH2 N-terminal amine and the influence of C-terminal modification on binding argue that the entire molecule (M-r 521) fits within P450 3A4. YPFP-NH2 was not oxidized by P450 3A4 but blocked binding of the substrates testosterone and midazolam, with K-i values similar to the spectral binding constant (K-s) for YPFP-NH2. YPFP-NH2 inhibited the oxidations of several typical P450 substrates with K-i values 10-fold greater than the K-s for binding YPFP-NH2 and its K-i for inhibiting substrate binding. The n values for cooperativity of these oxidations were not altered by YPFP-NH2, YPFP-NH2 inhibited the oxidations of midazolam at two different positions (1'- and 4-) with 20-fold different K-i values. The differences in the K-i values for blocking the binding to ferric P450 3A4 and the oxidation of several substrates may be attributed to weaker binding of YPFP-NH2 to ferrous P450 3A4 than to the ferric form. The ferrous protein can be considered a distinct form of the enzyme in binding and catalysis because many substrates (but not YPFP-NH2) facilitate reduction of the ferric to ferrous enzyme. Our results with these peptides are considered in the context of several proposed models. A P450 3A4 model based on these peptide studies contains at least two and probably three distinct ligand sites, with testosterone and alpha-naphthoflavone occupying distinct sites. Midazolam appears to be able to bind to P450 3A4 in two modes, one corresponding to the testosterone binding mode and one postulated to reflect binding in a third site, distinct from both testosterone and alpha-naphthoflavone, The work with indinavir and YPFP-NH2 also argues that room should be present in P450 3A4 to bind more than one smaller ligand in the "testosterone" site, although no direct evidence for such binding exists. Although this work with peptides provides evidence for the existence of multiple ligand binding sites, the results cannot be used to indicate their juxtaposition, which may vary through the catalytic cycle.