Human aromatase is a member of the cytochrome P450 superfamily, involved in steroid hormones biosynthesis. In particular, it converts androgen into estrogens being therefore responsible for the correct sex steroids balance. Due to its capacity in producing estrogens it has also been considered as a promising target for breast cancer therapy. Two single-nucleotide polymorphisms (R264C and R264H) have been shown to alter aromatase activity and they have been associated to an increased or decreased risk for estrogen-dependent pathologies. Here, the effect of these mutations on the protein dynamics is investigated by UV/FTIR and time resolved fluorescence spectroscopy. H/D exchange rates were measured by FTIR for the three proteins in the ligand-free, substrate- and inhibitor-bound forms and the data indicate that the wild-type enzyme undergoes a conformational change leading to a more compact tertiary structure upon substrate or inhibitor binding. Indeed, the H/D exchange rates are decreased when a ligand is present. In the variants, the exchange rates in the ligand-free and –bound forms are similar, indicating that a structural change is lacking, despite the single amino acid substitution is located in the peripheral shell of the protein molecule. Moreover, the fluorescence lifetimes data show that the quenching effect on tryptophan-224 observed upon ligand binding in the wild-type, is absent in both variants. Since this residue is located in the catalytic pocket, these findings suggest that substrate entrance and/or retention in the active site is partially compromised in both mutants. A contact network analysis demonstrates that the protein structure is organized in two main clusters, whose connectivity is altered by ligand binding, especially in correspondence of helix-G, where the amino acid substitutions occur. Our findings demonstrate that SNPs resulting in mutations on aromatase surface modify the protein flexibility that is required for substrate binding and catalysis. The cluster analysis provides a rationale for such effect, suggesting helix G as a possible target for aromatase inhibition.

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人类芳香酶的多态性影响蛋白质动力学和底物结合：光谱证据

人类芳香酶是细胞色素 P450 超家族的成员，参与类固醇激素的生物合成。特别是，它将雄激素转化为雌激素，因此负责正确的性类固醇平衡。由于其产生雌激素的能力，它也被认为是乳腺癌治疗的有希望的靶点。两种单核苷酸多态性（R264C 和 R264H）已被证明会改变芳香酶活性，并且它们与雌激素依赖性病变的风险增加或减少有关。在这里，通过 UV/FTIR 和时间分辨荧光光谱研究了这些突变对蛋白质动力学的影响。通过 FTIR 测量无配体中三种蛋白质的 H/D 交换率，底物和抑制剂结合形式和数据表明，野生型酶在底物或抑制剂结合时发生构象变化，导致更紧凑的三级结构。事实上，当存在配体时，H/D 交换率会降低。在变体中，无配体和结合形式的交换率相似，表明缺乏结构变化，尽管单个氨基酸取代位于蛋白质分子的外围壳中。此外，荧光寿命数据表明，在野生型配体结合时观察到的色氨酸 224 淬灭效应在两种变体中都不存在。由于该残基位于催化口袋中，这些发现表明底物进入和/或在活性位点中的保留在两种突变体中都部分受损。接触网络分析表明，蛋白质结构分为两个主要簇，其连接性因配体结合而改变，特别是在发生氨基酸取代的螺旋-G 的对应关系中。我们的研究结果表明，导致芳香酶表面突变的 SNP 改变了底物结合和催化所需的蛋白质灵活性。聚类分析为这种效应提供了基本原理，表明螺旋 G 可能是芳香化酶抑制的靶点。我们的研究结果表明，导致芳香酶表面突变的 SNP 改变了底物结合和催化所需的蛋白质灵活性。聚类分析为这种效应提供了基本原理，表明螺旋 G 可能是芳香化酶抑制的靶点。我们的研究结果表明，导致芳香酶表面突变的 SNP 改变了底物结合和催化所需的蛋白质灵活性。聚类分析为这种效应提供了基本原理，表明螺旋 G 可能是芳香化酶抑制的靶点。