Cléry C, Heiber-Langer I, Channac L, David L, Balny C, Masson P
Centre de Recherches du Service de Santé des Armées Emile Pardé, CRSSA, La Tronche, France.
Biochim Biophys Acta. 1995 Jul 3;1250(1):19-28. doi: 10.1016/0167-4838(95)00042-s.
Previous results on butyrylcholinesterase-catalyzed hydrolysis of o-nitrophenylbutyrate in the presence of soman, an irreversible inhibitor of cholinesterases, suggested that reversible binding of soman preceding enzyme phophonylation induced a new enzyme conformational state (E'). The purpose of the present study was to determine whether this effect depends on soman itself or is dependent on the presence and nature of substrate or ligand. First, we examined the effect of amiloride, a reversible cholinesterase effector, upon the butyrylcholinesterase-catalyzed hydrolysis of nitrophenyl esters. The effect of amiloride was found to be dependent on the position ortho or para of the substrate nitro group: amiloride acts as a non-linear reversible activator of p-nitrophenyl ester hydrolysis and as a non-linear reversible inhibitor of o-nitrophenyl ester hydrolysis. Second, the effect of amiloride upon hydrolysis of o/p-nitrophenylbutyrate was also studied under perturbing conditions, i.e., as a function of pressure (1-1600 bar) in the presence and absence of soman. Results show that the effect of reversible soman binding on butyrylcholinesterase activity in the presence of amiloride depends on the position of the substrate nitro group and amiloride concentration. Molecular modelling suggests that the presence of amiloride determines the orientation of ortho- and para-nitrophenyl esters in the active-site. gorge. The nitro group of o-nitrophenylbutyrate interacts with the oxyanion hole via hydrogen bonds and its phenyl ring interacts with amiloride whose heterocycle faces Trp-82. The nitro group of p-nitrophenylbutyrate does not interact with the oxyanion hole but points towards Tyr-332; the phenyl ring of p-nitrophenylbutyrate interacts with amiloride but there is no steric constraint on the acyl chain. Thus, the network of interactions in ternary complexes is tighter with o-nitrophenylbutryate as the substrate. There is no evidence for the existence of amiloride and/or soman-induced E' state when p-nitrophenylbutyrate is the substrate. On the other hand, reversible binding of amiloride and/or soman induces new active conformational states that may be either binary (or ternary) enzyme-ligand complex or new free enzyme conformation resulting from long-lived ligand-induced enzyme conformational change when o-nitrophenylbutyrate is the substrate. These ligand-induced states are stabilized by high pressure.
先前关于丁酰胆碱酯酶在胆碱酯酶不可逆抑制剂梭曼存在下催化邻硝基苯基丁酸酯水解的研究结果表明，梭曼在酶磷酸化之前的可逆结合诱导了一种新的酶构象状态（E'）。本研究的目的是确定这种效应是取决于梭曼本身，还是取决于底物或配体的存在及其性质。首先，我们研究了可逆性胆碱酯酶效应剂阿米洛利对丁酰胆碱酯酶催化的硝基苯基酯水解的影响。发现阿米洛利的效应取决于底物硝基的邻位或对位位置：阿米洛利对对硝基苯基酯水解起非线性可逆激活作用，而对对硝基苯基酯水解起非线性可逆抑制作用。其次，还在干扰条件下，即作为压力（1 - 1600巴）的函数，研究了阿米洛利在有和没有梭曼存在时对邻/对硝基苯基丁酸酯水解的影响。结果表明，在有阿米洛利存在时，梭曼的可逆结合对丁酰胆碱酯酶活性的影响取决于底物硝基的位置和阿米洛利的浓度。分子模拟表明，阿米洛利的存在决定了邻硝基和对硝基苯基酯在活性位点峡谷中的取向。邻硝基苯基丁酸酯的硝基通过氢键与氧负离子洞相互作用，其苯环与杂环面向色氨酸-82的阿米洛利相互作用。对硝基苯基丁酸酯的硝基不与氧负离子洞相互作用，而是指向酪氨酸-332；对硝基苯基丁酸酯的苯环与阿米洛利相互作用，但对酰基链没有空间限制。因此，以邻硝基苯基丁酸酯为底物时，三元复合物中的相互作用网络更紧密。当以对硝基苯基丁酸酯为底物时，没有证据表明存在阿米洛利和/或梭曼诱导的E'状态。另一方面，当以邻硝基苯基丁酸酯为底物时，阿米洛利和/或梭曼的可逆结合会诱导新的活性构象状态，这些状态可能是二元（或三元）酶-配体复合物，或者是由长寿命配体诱导的酶构象变化产生的新的游离酶构象。这些配体诱导的状态通过高压得以稳定。
