Laboratório de Modelação de Proteínas, ITQB-UNL, Av. da República, EAN, 2780-157 Oeiras, Portugal.
Protein Sci. 2011 Feb;20(2):379-86. doi: 10.1002/pro.569.
The phenomenon known as "ligand imprinting" or "ligand-induced enzyme memory" was first reported in 1988, when Russell and Klibanov observed that lyophilizing subtilisin in the presence of competitive inhibitors (that were subsequently removed) could significantly enhance its activity in an apolar solvent. (Russell and Klibanov, J Biol Chem 1988;263:11624-11626). They further observed that this enhancement did not occur when similar assays were carried out in water. Herein, we shed light on the molecular determinants of ligand imprinting using a molecular dynamics (MD) approach. To simulate the effect of placing an enzyme in the presence of a ligand before its lyophilization, an inhibitor was docked in the active site of subtilisin and 20 ns MD simulations in water were performed. The ligand was then removed and the resulting structure was used for subsequent MD runs using hexane and water as solvents. As a control, the same simulation setup was applied using the structure of subtilisin in the absence of the inhibitor. We observed that the ligand maintains the active site in an open conformation and that this configuration is retained after the removal of the inhibitor, when the simulations are carried out in hexane. In agreement with experimental findings, the structural configuration induced by the ligand is lost when the simulations take place in water. Our analysis of fluctuations indicates that this behavior is a result of the decreased flexibility displayed by enzymes in an apolar solvent, relatively to the aqueous situation.
“配体印迹”或“配体诱导的酶记忆”现象于 1988 年首次报道，当时 Russell 和 Klibanov 观察到，在存在竞争性抑制剂（随后被去除）的情况下冻干枯草杆菌蛋白酶，可以显著提高其在非极性溶剂中的活性。（Russell 和 Klibanov，J Biol Chem 1988；263:11624-11626）。他们进一步观察到，当在水中进行类似的测定时，这种增强不会发生。在这里，我们使用分子动力学（MD）方法阐明了配体印迹的分子决定因素。为了模拟在冻干前将酶置于配体存在下的效果，将抑制剂对接在枯草杆菌蛋白酶的活性部位，并在水中进行 20ns 的 MD 模拟。然后去除配体，并使用正己烷和水作为溶剂对所得结构进行后续 MD 运行。作为对照，在没有抑制剂的情况下，使用枯草杆菌蛋白酶的结构应用相同的模拟设置。我们观察到，配体保持了活性部位的开放构象，并且当在正己烷中进行模拟时，在去除抑制剂后，这种构象得以保留。与实验结果一致，当模拟在水中进行时，配体诱导的结构构型丢失。我们对波动的分析表明，这种行为是由于酶在非极性溶剂中相对水相显示出的柔韧性降低所致。
