Roston D, Cui Q
Theoretical Chemistry Institute, University of Wisconsin-Madison, Madison, WI, United States.
Methods Enzymol. 2016;577:213-50. doi: 10.1016/bs.mie.2016.05.016. Epub 2016 Jul 1.
Enzymology is approaching an era where many problems can benefit from computational studies. While ample challenges remain in quantitatively predicting behavior for many enzyme systems, the insights that often come from computations are an important asset for the enzymology community. Here we provide a primer for enzymologists on the types of calculations that are most useful for mechanistic problems in enzymology. In particular, we emphasize the integration of models that range from small active-site motifs to fully solvated enzyme systems for cross-validation and dissection of specific contributions from the enzyme environment. We then use a case study of the enzyme alkaline phosphatase to illustrate specific application of the methods. The case study involves examination of the binding modes of putative transition state analogues (tungstate and vanadate) to the enzyme. The computations predict covalent binding of these ions to the enzymatic nucleophile and that they adopt the trigonal bipyramidal geometry of the expected transition state. By comparing these structures with transition states found through free energy simulations, we assess the degree to which the transition state analogues mimic the true transition states. Technical issues worth treating with care as well as several remaining challenges to quantitative analysis of metalloenzymes are also highlighted during the discussion.
酶学正步入一个许多问题都能从计算研究中受益的时代。虽然在对许多酶系统的行为进行定量预测方面仍存在诸多挑战，但计算常常带来的见解对酶学界来说是一项重要财富。在此，我们为酶学研究者提供一份指南，介绍对酶学机理问题最有用的计算类型。特别地，我们强调从小的活性位点基序到完全溶剂化的酶系统等各种模型的整合，以进行交叉验证并剖析酶环境的特定贡献。然后，我们以碱性磷酸酶为例来说明这些方法的具体应用。该案例研究涉及考察假定的过渡态类似物（钨酸盐和钒酸盐）与酶的结合模式。计算预测这些离子与酶促亲核试剂发生共价结合，并且它们采用预期过渡态的三角双锥几何构型。通过将这些结构与通过自由能模拟找到的过渡态进行比较，我们评估过渡态类似物模拟真实过渡态的程度。讨论过程中还强调了值得谨慎对待的技术问题以及金属酶定量分析中仍存在的若干挑战。
