Neutron Structure of Human Carbonic Anhydrase II: Implications for Proton Transfer
Neutron Structure of Human Carbonic Anhydrase II: Implications for Proton Transfer
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DOI：
10.1021/bi901995n
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发表时间：
2010-01-26
期刊：
影响因子：
2.9
通讯作者：
Langan, Paul A.
中科院分区：
文献类型：
--
作者：
Fisher, S. Zoe;Kovalevsky, Andrey Y.;Langan, Paul A.
人碳酸酐酶II（HCA II）催化二氧化碳可逆水合形成碳酸氢盐和质子。尽管有许多高分辨率的X射线晶体结构，诱变和动力学数据，活性位点的结构细节，特别是质子转移途径，是不清楚的。在pH9.0条件下制备了HCA Ⅱ大晶体，并进行了H-D交换，用氘置换不稳定的氢。中子衍射研究在洛斯阿拉莫斯国家实验室的蛋白质晶体学站进行。2.0埃分辨率的结构揭示了几个有趣的活性部位特征：（1）Zn结合的溶剂似乎主要是D2 O分子，（2）活性位点空腔中溶剂分子的取向和氢键模式，（3）His 64的侧链未质子化（中性）并且主要处于指向锌的向内构象，（4）Tyr 7的酚侧链似乎是未质子化的。这些细节的影响进行了讨论，并提出了质子转移的建议机制。
Human carbonic anhydrase II (HCA II) catalyzes the reversible hydration of carbon dioxide to form bicarbonate and a proton. Despite many high-resolution X-ray crystal structures, mutagenesis, and kinetic data, the structural details of the active site, especially the proton transfer pathway,are unclear. A large HCA II crystal was prepared at pH 9.0 and Subjected to vapor H-Dexchange to replace labile hydrogens with deuteriums. Neutron diffraction studies were conducted at the Protein Crystallography Station at Los Alamos National Laboratory. The structure to 2.0 angstrom resolution reveals several interesting active site features: (1) the Zn-bound solvent appearing to be predominantly a D2O molecule, (2) the orientation and hydrogen bonding pattern of solvent molecules in the active site cavity, (3) the side chain of His64 being unprotonated (neutral) and predominantly in an inward conformation pointing toward the zinc, and (4) the phenolic side chain of Tyr7 appearing to be Unprotonated. The implications of these details are discussed, and a proposed mechanism for proton transfer is presented.