Substrate and Transition State Binding in Alkaline Phosphatase Analyzed by Computation of Oxygen Isotope Effects.
Substrate and Transition State Binding in Alkaline Phosphatase Analyzed by Computation of Oxygen Isotope Effects.
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DOI：
10.1021/jacs.6b07347
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发表时间：
2016-09-14
影响因子：
15
通讯作者：
Cui Q
中科院分区：
文献类型：
--
作者：
Roston D;Cui Q
酶是强大的催化剂，彻底了解其催化能力的来源将有助于许多医疗和工业应用。在这里，我们研究了碱性磷酸酶（AP）的催化机制，这是已知的最具催化活性的酶之一。我们已经使用量子力学计算和混合量子力学/分子力学（QM/MM）模拟模拟各种同位素效应相关的AP的反应。我们已经计算了平衡同位素效应（EIE），结合同位素效应（BIE），和动力学同位素效应（KIE）的范围内的磷酸单酯和二酯底物。结果与实验值吻合得很好，但反应过渡态（TS）的模型与这些实验的原始解释不同。我们的模型表明，同位素结合的影响作出重要贡献，测量KIEs的V/K，这复杂的解释测量值。我们的研究结果提供了一个详细的解释所测得的同位素效应，并作出预测，可以测试所提出的模型。该模型表明，在反应的基态（GS）的基板变形，部分类似于TS。高度预组织的活性位点优先结合类似于TS而不是GS的构象，这诱导底物适应酶，而不是与经典的“诱导适合”模型相反。TS相对于GS的优先稳定性降低了对化学步骤的屏障。
Enzymes are powerful catalysts and a thorough understanding of the sources of their catalytic power will facilitate many medical and industrial applications. Here we have studied the catalytic mechanism of alkaline phosphatase (AP), which is one of the most catalytically proficient enzymes known. We have used quantum mechanics calculations and hybrid quantum mechanics/molecular mechanics (QM/MM) simulations to model a variety of isotope effects relevant to the reaction of AP. We have calculated equilibrium isotope effects (EIEs), binding isotope effects (BIEs), and kinetic isotope effects (KIEs) for a range of phosphate mono- and diester substrates. The results agree well with experimental values, but the model for the reaction’s transition state (TS) differs from the original interpretation of those experiments. Our model indicates that isotope effects on binding make important contributions to measured KIEs on V/K, which complicated interpretation of the measured values. Our results provide a detailed interpretation of the measured isotope effects and make predictions that can test the proposed model. The model indicates that the substrate is deformed in the ground state (GS) of the reaction and partially resembles the TS. The highly preorganized active site preferentially binds conformations that resemble the TS and not the GS, which induces the substrate to adapt to the enzyme, rather than the other way around—as with classic “induced fit” models. The preferential stabilization of the TS over the GS is what lowers the barrier to the chemical step.
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影响因子：
15
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