Fisher S Zoë, Maupin C Mark, Budayova-Spano Monika, Govindasamy Lakshmanan, Tu Chingkuang, Agbandje-McKenna Mavis, Silverman David N, Voth Gregory A, McKenna Robert
Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, Florida 32610, USA.
Biochemistry. 2007 Mar 20;46(11):2930-7. doi: 10.1021/bi062066y. Epub 2007 Feb 24.
Human carbonic anhydrase II (HCA II) is a zinc-metalloenzyme that catalyzes the reversible interconversion of CO2 and HCO3-. The rate-limiting step of this catalysis is the transfer of a proton between the Zn-bound solvent molecule and residue His64. In order to fully characterize the active site structural features implicated in the proton transfer mechanism, the refined X-ray crystal structure of uncomplexed wild type HCA II to 1.05 A resolution with an Rcryst value of 12.0% and an Rfree value of 15.1% has been elucidated. This structure provides strong clues as to the pathway of the intramolecular proton transfer between the Zn-bound solvent and His64. The structure emphasizes the role of the solvent network, the unique positioning of solvent molecule W2, and the significance of the dual conformation of His64 in the active site. The structure is compared with molecular dynamics (MD) simulation calculations of the Zn-bound hydroxyl/His64+ (charged) and the Zn-bound water/His64 (uncharged) HCA II states. A comparison of the crystallographic anisotropic atomic thermal parameters and MD simulation root-mean-square fluctuation values show excellent agreement in the atomic motion observed between the two methods. It is also interesting that the observed active site solvent positions in the crystal structure are also the most probable positions of the solvent during the MD simulations. On the basis of the comparative study of the MD simulation results, the HCA II crystal structure observed is most likely in the Zn-bound water/His64 state. This conclusion is based on the following observations: His64 is mainly (80%) orientated in an inward conformation; electron density omit maps infer that His64 is not charged in an either inward or outward conformation; and the Zn-bound solvent is most likely a water molecule.
人碳酸酐酶II（HCA II）是一种锌金属酶，催化二氧化碳和碳酸氢根之间的可逆相互转化。该催化反应的限速步骤是锌结合溶剂分子与His64残基之间质子的转移。为了全面表征质子转移机制中涉及的活性位点结构特征，已阐明了未结合的野生型HCA II的X射线晶体结构，其分辨率达到1.05 Å，Rcryst值为12.0%，Rfree值为15.1%。该结构为锌结合溶剂与His64之间分子内质子转移途径提供了有力线索。该结构强调了溶剂网络的作用、溶剂分子W2的独特定位以及活性位点中His64双重构象的重要性。将该结构与锌结合羟基/His64 +（带电）和锌结合水/His64（不带电）HCA II状态的分子动力学（MD）模拟计算进行了比较。晶体学各向异性原子热参数与MD模拟均方根波动值的比较表明，两种方法观察到的原子运动具有极好的一致性。同样有趣的是，晶体结构中观察到的活性位点溶剂位置也是MD模拟过程中溶剂最可能的位置。基于对MD模拟结果的比较研究，观察到的HCA II晶体结构最可能处于锌结合水/His64状态。这一结论基于以下观察结果：His64主要（80%）呈向内构象；电子密度省略图推断His64在向内或向外构象中均不带电；并且锌结合溶剂最可能是水分子。
