Activation heat capacity is emerging as a crucial factor in enzyme thermoadaptation, as shown by the non-Arrhenius behaviour of many natural enzymes. However, its physical origin and relationship to the evolution of catalytic activity remain uncertain. Here we show that directed evolution of a computationally designed Kemp eliminase reshapes protein dynamics, which gives rise to an activation heat capacity absent in the original design. These changes buttress transition-state stabilization. Extensive molecular dynamics simulations show that evolution results in the closure of solvent-exposed loops and a better packing of the active site. Remarkably, this gives rise to a correlated dynamical network that involves the transition state and large parts of the protein. This network tightens the transition-state ensemble, which induces a negative activation heat capacity and non-linearity in the activity–temperature dependence. Our results have implications for understanding enzyme evolution and suggest that selectively targeting the conformational dynamics of the transition-state ensemble by design and evolution will expedite the creation of novel enzymes.

正如许多天然酶的非 Arrhenius 行为所示，活化热容量正在成为酶热适应中的一个关键因素。然而，它的物理起源和与催化活性演变的关系仍然不确定。在这里，我们展示了计算设计的 Kemp 消除酶的定向进化重塑了蛋白质动力学，从而产生了原始设计中不存在的活化热容量。这些变化支持过渡态稳定。广泛的分子动力学模拟表明，进化导致溶剂暴露环的闭合和活性位点的更好堆积。值得注意的是，这产生了一个涉及过渡态和大部分蛋白质的相关动态网络。这个网络收紧了过渡态集成，这会导致负活化热容量和活性 - 温度依赖性的非线性。我们的结果对理解酶的进化具有重要意义，并表明通过设计和进化选择性地针对过渡态整体的构象动力学将加速新型酶的产生。