QM/MM Free-Energy Simulations of Reaction in Serratia marcescens Chitinase B Reveal the Protonation State of Asp142 and the Critical Role of Tyr214
QM/MM Free-Energy Simulations of Reaction in Serratia marcescens Chitinase B Reveal the Protonation State of Asp142 and the Critical Role of Tyr214
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DOI：
10.1021/jp500652x
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发表时间：
2014-05-08
影响因子：
3.3
通讯作者：
Mulholland, Adrian J.
中科院分区：
文献类型：
--
作者：
Jitonnom, Jitrayut;Limb, Michael A. L.;Mulholland, Adrian J.
粘质沙雷氏菌几丁质酶B（ChiB）属于糖苷酶家族18（GH 18），通过一种不寻常的底物辅助机制催化β-1，4-糖苷键的水解，同时保持构型，其中底物本身充当分子内亲核试剂。在这里，两个基本步骤（糖基化和去糖基化）的ChiB催化的反应的研究通过结合量子力学/分子力学（QM/MM）伞形采样分子动力学（MD）模拟在SCC-DFTB/CHARMM 22理论水平。我们研究Asp 142质子化状态对反应的影响和该残留物在反应中的作用。我们的模拟表明，与中性天冬氨酸142的反应是优选的，并表明，该残基提供了在反应中形成的恶唑啉离子中间体的静电稳定。模拟还提供了对底物（结合在亚位点-1中）沿着优选反应途径所采用的构象路线（B-1，B-4 H-4（5）C-4（1））的深入了解。比较了用SCC-DFTB和B3 LYP方法计算的反应路径上沿着各驻点的相对能量。结果表明，SCC-DFTB是一种准确的方法，用于估计反应的两个步骤的相对势垒;然而，它被发现高估了反应中形成的中间体的相对能量时，与更高层次的理论相比。糖基化被认为是反应中的速率决定步骤，计算的总反应自由能垒为20.5千卡/摩尔，与来自实验的16.1千卡/摩尔势垒的合理协议。研究了Tyr 214在催化反应中的作用，结果表明该残基在反应的去糖基化步骤中起着关键作用。模拟的酶产物复合物也进行了一个未结合的事件建议已被观察到，提供潜在的新的机制洞察到产品的ChiB的释放。
Serratia marcescens Chitinase B (ChiB), belonging to the glycosidase family 18 (GH18), catalyzes the hydrolysis of beta-1,4-glycosidic bond, with retention of configuration, via an unusual substrate-assisted mechanism, in which the substrate itself acts as an intramolecular nucleophile. Here, both elementary steps (glycosylation and deglycosylation) of the ChiB-catalyzed reaction are investigated by means of combined quantum mechanics/molecular mechanics (QM/MM) umbrella sampling molecular dynamics (MD) simulations at the SCC-DFTB/CHARMM22 level of theory. We examine the influence of the Asp142 protonation state on the reaction and the role that this residue performs in the reaction. Our simulations show that reaction with a neutral Asp 142 is preferred and demonstrate that this residue provides electrostatic stabilization of the oxazolinium ion intermediate formed in the reaction. Insight into the conformational itinerary (B-1,B-4 H-4(5) C-4(1)) adopted by the substrate (bound in subsite -1) along the preferred reaction pathway is also provided by the simulations. The relative energies of the stationary points found along the reaction pathway calculated with SCC-DFTB and B3LYP were compared. The results suggest that SCC-DFTB is an accurate method for estimating the relative barriers for both steps of the reaction; however, it was found to overestimate the relative energy of an intermediate formed in the reaction when compared with the higher level of theory. Glycosylation is suggested to be a rate-determining step in the reaction with calculated overall reaction free-energy barrier of 20.5 kcal/mol, in a reasonable agreement with the 16.1 kcal/mol barrier derived from the experiment. The role of Tyr214 in catalysis was also investigated with the results, indicating that the residue plays a critical role in the deglycosylation step of the reaction. Simulations of the enzyme-product complex were also performed with an unbinding event suggested to have been observed, affording potential new mechanistic insight into the release of the product of ChiB.