Department of Physics and Astronomy, University College London, London WC1E 6BT, United Kingdom.
J Am Chem Soc. 2011 Mar 16;133(10):3548-56. doi: 10.1021/ja109712q. Epub 2011 Feb 22.
Hydrogenases are enzymes that catalyze the reversible conversion of hydrogen molecules to protons and electrons. The mechanism by which the gas molecules reach the active site is important for understanding the function of the enzyme and may play a role in the selectivity for hydrogen over inhibitor molecules. Here, we develop a general multiscale molecular simulation approach for the calculation of diffusion rates and determination of pathways by which substrate or inhibitor gases can reach the protein active site. Combining kinetic data from both equilibrium simulations and enhanced sampling, we construct a master equation describing the movement of gas molecules within the enzyme. We find that the time-dependent gas population of the active site can be fit to the same phenomenological rate law used to interpret experiments, with corresponding diffusion rates in very good agreement with experimental data. However, in contrast to the conventional picture, in which the gases follow a well-defined hydrophobic tunnel, we find that there is a diverse network of accessible pathways by which the gas molecules can reach the active site. The previously identified tunnel accounts for only about 60% of the total flux. Our results suggest that the dramatic decrease in the diffusion rate for mutations involving the residue Val74 could be in part due to the narrowing of the passage Val74-Arg476, immediately adjacent to the binding site, explaining why mutations of Leu122 had only a negligible effect in experiment. Our method is not specific to the [NiFe]-hydrogenase and should be generally applicable to the transport of small molecules in proteins.
氢化酶是一种能够催化氢气分子可逆转化为质子和电子的酶。气体分子到达活性位点的机制对于理解酶的功能很重要，并且可能对酶对氢气和抑制剂分子的选择性起作用。在这里，我们开发了一种通用的多尺度分子模拟方法，用于计算扩散率，并确定底物或抑制剂气体到达蛋白质活性位点的途径。通过结合来自平衡模拟和增强采样的动力学数据，我们构建了一个描述气体分子在酶内运动的主方程。我们发现，活性位点的气体时变种群可以拟合到用于解释实验的相同唯象速率定律，相应的扩散率与实验数据非常吻合。然而，与传统观点相反，在传统观点中，气体遵循明确的疏水性隧道，我们发现气体到达活性位点有多种可及的途径。先前确定的隧道仅占总通量的约 60%。我们的结果表明，涉及残基 Val74 的突变导致扩散率急剧下降，部分原因可能是由于紧邻结合位点的 Val74-Arg476 通道变窄，这解释了为什么 Leu122 的突变在实验中几乎没有影响。我们的方法不仅限于[NiFe]-氢化酶，并且应该普遍适用于蛋白质中小分子的运输。
