Free Energy Simulations of Active-Site Mutants of Dihydrofolate Reductase
Free Energy Simulations of Active-Site Mutants of Dihydrofolate Reductase
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DOI：
10.1021/jp5059963
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发表时间：
2015-01-22
影响因子：
3.3
通讯作者：
Major, Dan Thomas
中科院分区：
文献类型：
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作者：
Doron, Dvir;Stojkovic, Vanja;Major, Dan Thomas
本研究采用混合量子力学/分子力学（QM/MM）模拟研究了大肠杆菌二氢叶酸还原酶（DHFR）活性位点残基114突变对氢化物转移的影响。最近对114X突变体（X = V， A和G）的动力学测量表明，随着114侧链的系统性减少，氢化物转移速度变慢，动力学同位素效应（KlEs）越来越依赖于温度。QM/MM模拟表明，当原始异亮氨酸残基在硅中被缬氨酸、丙氨酸或甘氨酸（分别为I14V、I14A和I14G DHFR）取代时，氢化物转移反应的自由能垒-高度相对于野生型酶增加。这些趋势与这些系统报告的单次周转率测量结果一致。此外，对反应性Michaelis复合体的扩展动力学模拟显示，突变体（尤其是I14G突变体）的灵活性增强，包括与天然酶相比，don-Or受体距离（DAD）和活性位点氢键网络有相当大的波动。这些观察结果表明，突变引起的扰动部分损害了反应物状态下的活性位点环境。另一方面，所有DHER变体的过渡状态的平均父亲是相似的。114个突变体（V， A）的晶体结构证实了M20和其他环的柔韧性增加的趋势。
This study employs hybrid quantum mechanics molecular-mechanics (QM/MM) simulations to investigate the effect of mutations of the active-site residue 114 of E. coli dihydrofolate reductase (DHFR) on the hydride transfer. Recent kinetic measurements of the 114X mutants (X = V; A, and G) indicated slower hydride transfer fates and increasingly temperature-dependent kinetic isotope effects (KlEs) withsystematic reduction of the 114 side chain. The QM/MM simulations snow that when the original isoleucine residue is substituted in silico by valine, alanine, or glycine (I14V, I14A, and I14G DHFR, respectively), the free energy barrier- height of the hydride transfer, reaction increases relative to the wild-type-enzyme. These trends are in line with the single-turnover rate measurements reported for these systems. In addition, extended dynamics simulations of the reactive Michaelis complex reveal enhanced flexibility in the mutants, and in particular for the I14G mutant, including considerable fluctuations of the don-Or acceptor distance (DAD) and the active-sitehydrogen bonding network compared with those detected in the native enzyme. These 'observations suggest that the perturbations induced by the mutations partly impair the actiye-site environment in the reactant state. On the other hand, the average DADs at the transition state of all DHER variants are similar. Crystal structures of 114 Mutants,(V, A, and confirmed the trend of increased flexibility of the M20 and other loops.