Extensive molecular dynamics (MD) simulations on designed and evolved enzymes for the Kemp Elimination were performed. Over thirty different systems including combinations of three protein scaffolds used in computational design (KE70, KE59 and HG3), several directed evolution variants and ligands (5‐nitro‐benzoisoxazole, and 5‐nitro‐benzotriazole), were screened computationally. The study was focused mostly on computational and evolved variants for which X‐ray structures are available. MD simulations were used to monitor the organization of the active sites over time periods of 100 ns. A general trend was observed in the simulations: the optimal organization of active site residues, corresponding to the most active catalysts, is only maintained with polarized, TS‐like substrates, reinforcing the crucial role of protein dynamics and electrostatic preorganization for efficient biocatalysis. Implications for enzyme design protocols are discussed.

中文翻译：

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进化的 Kemp 消除酶中底物极性和蛋白质动力学之间的相互作用

对为坎普消除而设计和进化的酶进行了广泛的分子动力学 (MD) 模拟。通过计算筛选了三十多个不同的系统，包括计算设计中使用的三种蛋白质支架的组合（KE70、KE59和HG3）、几种定向进化变体和配体（5-硝基苯并异恶唑和5-硝基苯并三唑）。这项研究主要集中在 X 射线结构可用的计算和进化变体上。 MD 模拟用于监测 100 ns 时间内活性位点的组织。在模拟中观察到了一个总体趋势：对应于最活跃的催化剂的活性位点残基的最佳组织仅通过极化的、类似 TS 的底物来维持，这增强了蛋白质动力学和静电预组织对于高效生物催化的关键作用。讨论了酶设计方案的影响。