Mechanistic investigation of sulfonamide ligands as human carbonic anhydrase II inhibitors.
Mechanistic investigation of sulfonamide ligands as human carbonic anhydrase II inhibitors.
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DOI：
10.1016/j.ijbiomac.2018.08.186
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发表时间：
2018-12
影响因子：
8.2
通讯作者：
L. Alaei;R. Khodarahmi;V. Sheikh-hasani;N. Sheibani;A. Moosavi-Movahedi
中科院分区：
文献类型：
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作者：
L. Alaei;R. Khodarahmi;V. Sheikh-hasani;N. Sheibani;A. Moosavi-Movahedi
利用紫外-可见光谱、荧光光谱、圆二色谱和分子动力学模拟等技术研究了磺酰胺类配体对人碳酸酐酶II（HCA II）结构和功能的影响。在27 °C下在50 mM Tris-HCl，pH 7.4中进行动力学测量。动力学数据显示，磺酰胺配体抑制HCA II酯酶活性的线性竞争方式与Ki在纳摩尔范围内。荧光测量表明，配体作为酶猝灭剂。在不同温度下的猝灭数据的Stern-Volmer分析表明，通过静态和动态猝灭机制发生的HCA II的固有荧光猝灭。结合热力学参数分析表明，氢键和疏水相互作用在酶-药物复合物的稳定性中起着重要作用。Job图证实了配体-蛋白质复合物的1：1化学计量比，因此，存在一个配体结合位点。分子模拟证实，乙酰唑胺诱导的两个结构域相邻的活性位点，包括氨基酸19-25和61-67令人印象深刻的构象变化。RMSF研究表明，药物结合后，活性位点附近的三个不同区域（包括氨基酸15-25、160-180和190-210）发生急剧变化。
The effect of some sulfonamide ligands on the structure and function of human carbonic anhydrase II (HCA II) was investigated using different spectroscopic techniques including UV–Vis, fluorescence, circular dichroism and molecular dynamics simulation tools. Kinetic measurements were performed in 50 mM Tris-HCl, pH 7.4 at 27 °C. Kinetic data revealed that sulfonamide ligands inhibit the HCA II esterase activity in a linear competitive manner with Kiin the nanomolar range. Fluorescence measurements illustrated that ligands act as the enzyme quenchers. Stern–Volmer analysis of the quenching data at different temperatures demonstrated that the quenching of the HCA II intrinsic fluorescence occurred through static and dynamic quenching mechanisms. Analysis of the binding thermodynamic parameters showed that hydrogen bonding and hydrophobic interactions play an important role in the stabilization of enzyme–drug complex. Job plot confirmed the 1:1 stoichiometry of ligand-protein complex, and therefore, the existence of one binding site for the ligand. Molecular simulations confirmed that acetazolamide induced impressive conformational changes in two domains adjacent to the active site, including amino acids 19–25 and 61–67. RMSF studies showed sharp changes in three distinct regions near the active site including amino acids 15–25, 160–180 and 190–210 upon drug binding.