Jitonnom Jitrayut, Limb Michael A L, Mulholland Adrian J
Division of Chemistry, School of Science, University of Phayao , Phayao 56000, Thailand.
J Phys Chem B. 2014 May 8;118(18):4771-83. doi: 10.1021/jp500652x. Epub 2014 Apr 29.
Serratia marcescens Chitinase B (ChiB), belonging to the glycosidase family 18 (GH18), catalyzes the hydrolysis of β-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 Asp142 is preferred and demonstrate that this residue provides electrostatic stabilization of the oxazolinium ion intermediate formed in the reaction. Insight into the conformational itinerary ((1,4)B↔(4)H5↔(4)C1) 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.
粘质沙雷氏菌几丁质酶B（ChiB）属于糖苷酶家族18（GH18），它通过一种不同寻常的底物辅助机制催化β-1,4-糖苷键的水解反应，并保留其构型，在该机制中底物自身充当分子内亲核试剂。在此，通过在SCC-DFTB/CHARMM22理论水平下结合量子力学/分子力学（QM/MM）伞形采样分子动力学（MD）模拟，对ChiB催化反应的两个基本步骤（糖基化和去糖基化）进行了研究。我们考察了Asp142质子化状态对反应的影响以及该残基在反应中所起的作用。我们的模拟表明与中性Asp142的反应更受青睐，并证明该残基为反应中形成的恶唑啉离子中间体提供静电稳定作用。模拟还提供了对底物（结合在亚位点-1）沿着优选反应途径所采用的构象路径（(1,4)B↔(4)H5↔(4)C1）的深入了解。比较了用SCC-DFTB和B3LYP计算得出的沿着反应途径找到的驻点的相对能量。结果表明，SCC-DFTB是估计反应两个步骤相对势垒的准确方法；然而，与更高水平的理论相比，发现它高估了反应中形成的中间体的相对能量。糖基化被认为是反应中的速率决定步骤，计算得出的总反应自由能垒为20.5 kcal/mol，与实验得出的16.1 kcal/mol的势垒合理相符。还对Tyr214在催化中的作用进行了研究，结果表明该残基在反应的去糖基化步骤中起关键作用。还对酶-产物复合物进行了模拟，并提出观察到了解离事件，为ChiB产物的释放提供了潜在的新机制见解。
