Location of binding sites in small molecule rescue of human carbonic anhydrase II
Location of binding sites in small molecule rescue of human carbonic anhydrase II
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DOI：
10.1529/biophysj.106.093203
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发表时间：
2007-01-01
影响因子：
3.4
通讯作者：
Silverman, David N.
中科院分区：
文献类型：
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作者：
Bhatt, Deepa;Fisher, S. Zoe;Silverman, David N.
人碳酸酐酶II （HCA II）突变体的小分子拯救是通过外源性供体/受体参与锌结合水和溶液之间的质子转移途径发生的。为了更彻底地研究这种激活的能量学，我们构建了一个突变体H64W HCA II，我们已经证明它被4-甲基咪唑（4-MI）激活，其机制涉及4-MI与Trp-64侧链的结合，类似于锌的8埃。一系列实验结果表明，作为外源质子给体的咪唑和吡啶衍生物与Trp-64的吲哚环相互作用可以激活H64W - HCA II；这些实验包括与质子转移相一致的pH谱和H/D溶剂同位素效应，观察到与Gly-64相比，含有Trp-64的突变体的激活大约增加了四倍，以及通过x射线晶体学观察到W5A-H64W HCA II中与Trp-64的吲哚侧链相关的4-MI结合。在W5A-H64W HCA II中，结合在较不灵活的Trp-64侧链上的质子供体没有表现出活化，但这些结合在H64W HCA II中较灵活的Trp-64侧链上的质子供体确实表现出活化，这支持了结合位点的构象迁移与更有效的质子转移有关的建议。利用Marcus理论进行评价表明，这些质子供体对H64W HCA II的活化反映在功函数w(r)和w(p)上，而不是在本征Marcus势垒本身上，这与溶剂重组在催化中的作用是一致的。
Small molecule rescue of mutant forms of human carbonic anhydrase II (HCA II) occurs by participation of exogenous donors/acceptors in the proton transfer pathway between the zinc-bound water and solution. To examine more thoroughly the energetics of this activation, we have constructed a mutant, H64W HCA II, which we have shown is activated by 4-methylimidazole (4-MI) by a mechanism involving the binding of 4-MI to the side chain of Trp-64 similar to 8 angstrom from the zinc. A series of experiments are consistent with the activation of H64W HCA II by the interaction of imidazole and pyridine derivatives as exogenous proton donors with the indole ring of Trp-64; these experiments include pH profiles and H/D solvent isotope effects consistent with proton transfer, observation of approximately fourfold greater activation with the mutant containing Trp-64 compared with Gly-64, and the observation by x-ray crystallography of the binding of 4-MI associated with the indole side chain of Trp-64 in W5A-H64W HCA II. Proton donors bound at the less flexible side chain of Trp-64 in W5A-H64W HCA II do not show activation, but such donors bound at the more flexible Trp-64 of H64W HCA II do show activation, supporting suggestions that conformational mobility of the binding site is associated with more efficient proton transfer. Evaluation using Marcus theory showed that the activation of H64W HCA II by these proton donors was reflected in the work functions w(r) and w(p) rather than in the intrinsic Marcus barrier itself, consistent with the role of solvent reorganization in catalysis.