Kinetic isotope effects for concerted multiple proton transfer:: A direct dynamics study of an active-site model of carbonic anhydrase
Kinetic isotope effects for concerted multiple proton transfer:: A direct dynamics study of an active-site model of carbonic anhydrase
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DOI：
10.1021/ja0210594
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发表时间：
2003-01-08
影响因子：
15
通讯作者：
Cui, Q
中科院分区：
文献类型：
--
作者：
Smedarchina, Z;Siebrand, W;Cui, Q
由酶碳酸酐酶II，从体液中除去二氧化碳，催化的反应的速率常数计算的活性位点的模型。速率决定步骤是通过两个或更多个水分子的桥将质子从锌结合的水分子转移到组氨酸残基。除了水分子的数量和位置之外，活性位点的结构从X射线研究中已知。模型计算报告为58个原子的系统，包括一个四配位的锌离子连接到一个甲基咪唑分子的两个沃茨链，约束复制的活性位点的大小。在自洽电荷密度泛函紧束缚（SCC-DFTB）水平下，通过质子转移步骤的近似密度泛函处理计算结构和振动力场。发现一个单一的过渡态，表明协同三重质子转移。基于近似瞬子方法和变分过渡态理论与Tunnel修正的质子和氘核转移及其组合的直接动力学计算是相当一致的，产率相当高，动力学同位素效应（KIES）略高于实验。从过渡态理论得到的经典速率常数小于量子值，但相应的KIES大5倍。对于多个质子转移沿着水桥的经典KIES被示出通常大于量子KIES，这使区分隧穿和过势垒转移的标准方法失效。在目前的情况下，一个三向比较的经典和量子结果与观察到的数据是必要的，以得出结论，质子转移沿着的桥梁进行隧道。结果表明，两个水桥是存在于低浓度，但由于其高效率的质子传输作出了重大贡献。含有更多水分子的桥接结构可能具有较低的能量，但预计效率较低。所观察到的指数依赖性的KIES的氘浓度在H2O/D2 O混合物意味着协同转移，从而排除了大量的贡献，导致逐步转移通过溶剂化的水合氢离子，这大概占主导地位的质子转移效率较低的碳酸酐酶同工酶的结构。
The rate constant of the reaction catalyzed by the enzyme carbonic anhydrase II, which removes carbon dioxide from body fluids, is calculated for a model of the active site. The rate-determining step is proton transfer from a zinc-bound water molecule to a histidine residue via a bridge of two or more water molecules. The structure of the active site is known from X-ray studies except for the number and location of the water molecules. Model calculations are reported for a system of 58 atoms including a four-coordinated zinc ion connected to a methylimidazole molecule by a chain of two waters, constrained to reproduce the size of the active site. The structure and vibrational force field are calculated by an approximate density functional treatment of the proton-transfer step at the Self-Consistent-Charge Density Functional Tight Binding (SCC-DFTB) level. A single transition state is found indicating concerted triple proton transfer. Direct-dynamics calculations for proton and deuteron transfer and combinations thereof, based on the Approximate Instanton Method and on Variational Transition State Theory with Tunneling Corrections, are in fair agreement and yield rates that are considerably higher and kinetic isotope effects (KIES) that are somewhat higher than experiment. Classical rate constants obtained from Transition State Theory are smaller than the quantum values but the corresponding KIES are five times larger. For multiple proton transfer along water bridges classical KIES are shown to be generally larger than quantum KIES, which invalidates the standard method to distinguish tunneling and over-barrier transfer. In the present case, a three-way comparison of classical and quantum results with the observed data is necessary to conclude that proton transfer along the bridge proceeds by tunneling. The results suggest that the two-water bridge is present in low concentrations but makes a substantial contribution to proton transport because of its high efficiency. Bridging structures containing more water molecules may have lower energies but are expected to be less efficient. The observed exponential dependence of the KIES on the deuterium concentration in H2O/D2O mixtures implies concerted transfer and thus rules out substantial contributions from structures that lead to stepwise transfer via solvated hydronium ions, which presumably dominate proton transfer in less efficient carbonic anhydrase isozymes.