Dissection of the pH dependence of inhibitor binding energetics for an aspartic protease: direct measurement of the protonation states of the catalytic aspartic acid residues.
Dissection of the pH dependence of inhibitor binding energetics for an aspartic protease: direct measurement of the protonation states of the catalytic aspartic acid residues.
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天冬氨酸蛋白酶抑制剂结合能量的 pH 依赖性剖析：直接测量催化天冬氨酸残基的质子化状态。
DOI：
10.1021/bi971550l
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发表时间：
1997
期刊：
影响因子：
2.9
通讯作者：
Erickson,JW
中科院分区：
文献类型：
--
作者：
Xie,D;Gulnik,S;Collins,L;Gustchina,E;Suvorov,L;Erickson,JW
酶的催化活性和抑制剂结合能通常是pH依赖性的。天冬氨酸蛋白酶是一类重要的基于结构的药物设计的酶靶点。我们已经进行了一个完整的热力学研究胃蛋白酶抑制剂结合plasmepsin II，天冬氨酸蛋白酶发现在恶性疟原虫，使用等温滴定量热法和圆二色性。热力学参数（ΔG、ΔH、ΔCp和ΔS）作为pH和温度的函数被测量。在4.5至7.0的pH范围内，胃蛋白酶抑制剂结合伴随着溶剂和复合物之间的质子转移。我们使用热力学质子键理论推导出胃酶抑素的pH独立的结合能和可电离残基的数量和pKa值，其pKa值在配体结合过程中发生变化。根据胃蛋白酶抑制剂-血浆蛋白酶II复合物的三维结构，这些残基被鉴定为两个催化性的组氨酸，pKas为6.5和3.0，His 164，pKas为7.5。在pH 5.0时，蛋白酶具有最佳活性，质子转移过程贡献了总结合自由能变化的近40%，活性位点天冬氨酸残基的总电荷为-1。这些实验结果提供了直接测量的质子化状态的催化剂在结合配体的存在下。胃蛋白酶抑制剂与人组织蛋白酶D，溶酶体天冬氨酸蛋白酶，共享35%的序列同一性与plasmepsin II结合的热力学和结构数据的比较表明，这两种蛋白质之间的能量差异是由于更高的结构域间的灵活性plasmepsin II。
The catalytic activity and inhibitor binding energetics of enzymes are often pH-dependent properties. Aspartic proteases comprise an important class of enzyme targets for structure-based drug design. We have performed a complete thermodynamic study of pepstatin binding to plasmepsin II, an aspartic proteinase found inPlasmodium falciparum, using isothermal titration calorimetry and circular dichroism. Thermodynamic parameters (ΔG, ΔH, ΔCp, and ΔS) were measured as functions of both pH and temperature. In the pH range from 4.5 to 7.0, pepstatin binding is accompanied by proton transfer between the solvent and the complex. We used thermodynamic proton linkage theory to derive both the pH-independent binding energetics for pepstatin and the number and pKavalues of ionizable residues whose pKavalues change during ligand binding. These residues were identified as the two catalytic aspartates, with pKas of 6.5 and 3.0, and His 164, with a pKaof 7.5, based on the three-dimensional structure of the pepstatin−plasmepsin II complex. At pH 5.0, where the protease has optimum activity, the proton transfer process contributes almost 40% of the total binding free energy change and the total charge of the active-site aspartic acid residues is −1. These experimental results provide direct measurement for the protonation states of the catalytic aspartates in the presence of bound ligands. Comparison of the thermodynamic and structural data for pepstatin binding with human cathepsin D, a lysosomal aspartic protease that shares 35% sequence identity with plasmepsin II, suggests that the energetic differences between these two proteins are due to a higher interdomain flexibility in plasmepsin II.