Predicting nitrogen and oxygen kinetic isotope effects of nitrate reduction by periplasmic dissimilatory nitrate reductase
Predicting nitrogen and oxygen kinetic isotope effects of nitrate reduction by periplasmic dissimilatory nitrate reductase
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预测周质异化硝酸盐还原酶还原硝酸盐的氮和氧动力学同位素效应
DOI：
10.1016/j.gca.2020.10.027
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发表时间：
2021-01-15
影响因子：
5
通讯作者：
Liu，Yun
中科院分区：
文献类型：
--
作者：
He，Yuyang;Zhang，Yining;Liu，Yun
动力学同位素效应（KIE）揭示了基元反应的过渡态结构，并可以通过量子化学计算进行预测。用密度泛函理论计算大量原子的酶促反应在计算上是不允许的，特别是如果考虑明确的溶剂效应。截断法将整个分子简化为一个围绕目标位置的团簇，可以简化有机大分子位置特异性平衡同位素效应的计算。它也应该适用于KIE计算的酶反应，并允许我们引入明确的溶剂分子的系统。如果这种处理方法是可行的和可靠的，它将提供一个有效的方法来估计由酶反应产生的KIE的数量。显然，必须测试其鲁棒性。本文以Rhodobacter sphaeroides的周质异化硝酸还原酶（Nap）活性位点还原NO3−为例，建立了17个模型，分别考察了截断值、隐式和显式溶剂效应对KIE计算的影响。结果表明，为了准确、有效地估算酶促反应的KIE值，可以将反应模型简化为在活性位附近有3个键的截断模型。然后，结合隐式加显式溶剂模型可以更真实地模拟反应环境，这是准确性所必需的。我们计算的25 °C下硝酸盐Nap还原的lnKIE值分别为-32.4 ± 1.8‰（15 N）和-20.9 ± 0.4 ‰（18 O），ln 18 KIE/ln 15 KIE比值为0.65 ± 0.05。虽然需要考虑额外的NO3-输运过程，但我们的计算结果与实验室实验中校准的同位素效应一致，表明我们计算的过渡态描述了Nap还原NO3−的一般反应机制。
Kinetic isotope effect (KIE) reveals the transition state structure of an elementary reaction and can be predicted by quantum chemical calculation. Density Functional Theory calculation of an enzymatic reaction with large numbers of atoms is computationally prohibitive, especially if explicit solvent effect is considered. Cutoff method, which simplifies an entire molecule to a cluster around a target position, can simplify position-specific equilibrium isotope effect calculation for a large organic molecule. It should also be applicable to KIE calculation of an enzymatic reaction and allow us to introduce explicit solvent molecules to the system. If this treatment is feasible and trustable, it will provide an efficient method to estimate a number of KIEs produced by enzyme reactions. Obviously, its robustness must be tested. Here, using NO3−reduction by the active site of periplasmic dissimilatory nitrate reductase (Nap) inRhodobacter sphaeroidesas an example, we built 17 models to test the influence of cutoff size, implicit, and explicit solvent effects on KIE calculation. The results show that to estimate the KIE value of an enzymatic reaction accurately and efficiently, we can first simplify the reaction model to a cutoff model with 3 proximal bonds to the active position. Then, incorporating implicit-plus-explicit solvent models can simulate a reaction environment more realistically, which is necessary for accuracy. Our calculated lnKIEvalues for nitrate Nap reduction at 25 °C are −32.4 ± 1.8‰ for15N and −20.9 ± 0.4 ‰ for18O, respectively, with a ln18KIE/ln15KIEratio of 0.65 ± 0.05. Although additional reservoir-transport processes need to be considered, our calculation results are consistent with calibrated isotope effects from laboratory experiments, suggesting that the transition state we calculated depicts the general reaction mechanism of NO3−reduction by Nap.