Hu Hao, Boone Amy, Yang Weitao
Department of Chemistry, Duke University, Durham, North Carolina 27708, USA.
J Am Chem Soc. 2008 Nov 5;130(44):14493-503. doi: 10.1021/ja801202j. Epub 2008 Oct 8.
Despite extensive experimental and theoretical studies, the detailed catalytic mechanism of orotidine 5'-monophosphate decarboxylase (ODCase) remains controversial. In particular simulation studies using high level quantum mechanics have failed to reproduce experimental activation free energy. One common feature of many previous simulations is that there is a water molecule in the vicinity of the leaving CO2 group whose presence was only observed in the inhibitor bound complex of ODCase/BMP. Various roles have even been proposed for this water molecule from the perspective of stabilizing the transition state and/or intermediate state. We hypothesize that this water molecule is not present in the active ODCase/OMP complex. Based on QM/MM minimum free energy path simulations with accurate density functional methods, we show here that in the absence of this water molecule the enzyme functions through a simple direct decarboxylation mechanism. Analysis of the interactions in the active site indicates multiple factors contributing to the catalysis, including the fine-tuned electrostatic environment of the active site and multiple hydrogen-bonding interactions. To understand better the interactions between the enzyme and the inhibitor BMP molecule, simulations were also carried out to determine the binding free energy of this special water molecule in the ODCase/BMP complex. The results indicate that the water molecule in the active site plays a significant role in the binding of BMP by contributing approximately -3 kcal/mol to the binding free energy of the complex. Therefore, the complex of BMP plus a water molecule, instead of the BMP molecule alone, better represents the tight binding transition state analogue of ODCase. Our simulation results support the direct decarboxylation mechanism and highlight the importance of proper recognition of protein bound water molecules in the protein-ligand binding and the enzyme catalysis.
尽管进行了广泛的实验和理论研究，但乳清苷5'-单磷酸脱羧酶（ODCase）的详细催化机制仍存在争议。特别是使用高水平量子力学的模拟研究未能重现实验活化自由能。许多先前模拟的一个共同特征是，在离去的CO2基团附近存在一个水分子，而这种水分子仅在ODCase/BMP的抑制剂结合复合物中观察到。从稳定过渡态和/或中间态的角度，甚至对该水分子提出了各种作用。我们假设在活性ODCase/OMP复合物中不存在这种水分子。基于使用精确密度泛函方法的QM/MM最小自由能路径模拟，我们在此表明，在没有这种水分子的情况下，酶通过简单的直接脱羧机制发挥作用。对活性位点相互作用的分析表明，有多种因素有助于催化，包括活性位点微调的静电环境和多种氢键相互作用。为了更好地理解酶与抑制剂BMP分子之间的相互作用，还进行了模拟以确定该特殊水分子在ODCase/BMP复合物中的结合自由能。结果表明，活性位点中的水分子通过对复合物的结合自由能贡献约-3 kcal/mol，在BMP的结合中起重要作用。因此，BMP加上一个水分子的复合物，而不是单独的BMP分子，更好地代表了ODCase的紧密结合过渡态类似物。我们的模拟结果支持直接脱羧机制，并突出了在蛋白质-配体结合和酶催化中正确识别蛋白质结合水分子的重要性。
