Aqvist J, Fothergill M
Department of Molecular Biology, Uppsala University, Biomedical Centre, Box 590, S-75124 Uppsala, Sweden.
J Biol Chem. 1996 Apr 26;271(17):10010-6. doi: 10.1074/jbc.271.17.10010.
A major challenge for theoretical simulation methods is the calculation of enzymic reaction rates directly from the three-dimensional protein structure together with some idea of the chemical reaction mechanism. Here, we report the evaluation of a complete free energy profile for all the elementary steps of the triosephosphate isomerase catalyzed reaction using such an approach. The results are compatible with available experimental data and also suggest which of the possible reaction intermediates is kinetically observable. In addition to previously identified catalytic residues, the simulations show that a crystallographically observed active site water molecule plays an important role during catalysis and an intersubunit interaction that could explain the low activity of the monomeric enzyme is also observed. The calculations clearly demonstrate the important catalytic effects associated with stabilization of charged high energy intermediates and reduction of reorganization energy, which are likely to be general principles of enzyme catalyzed charge transfer and separation reactions.
理论模拟方法面临的一个主要挑战是如何根据三维蛋白质结构以及化学反应机制的一些概念直接计算酶促反应速率。在此，我们报告了使用这种方法对磷酸丙糖异构酶催化反应的所有基本步骤进行完整自由能分布评估的结果。这些结果与现有的实验数据相符，还表明了哪些可能的反应中间体在动力学上是可观测的。除了先前确定的催化残基外，模拟结果表明，晶体学观察到的活性位点水分子在催化过程中起着重要作用，并且还观察到一种亚基间相互作用，这可以解释单体酶活性较低的原因。计算结果清楚地证明了与带电高能中间体的稳定化以及重组能的降低相关的重要催化作用，这可能是酶催化电荷转移和分离反应的一般原理。
