底物及抑制剂对重组在人工磷脂膜上的乙酰胆碱酯酶四聚体亚分子结构的影响
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摘要
目的:为探索乙酰胆碱酯酶(AChE)水解底物强大功能的可能机制,观察底物及外周阴离子位点(PAS)抑制剂对重组在云母表面人工磷脂膜上的AChE四聚体(AChE G4)亚分子结构的影响。内容:重组于云母表面的人工磷脂膜的制备;AChE在人工磷脂膜上的重组;AChE多分子型的形态学研究;AChE G4和乙酰胆碱酯酶单体(AChE G1)的分离及纯化;ACh及抑制剂对重组于云母表面人工磷脂膜上的AChE G4结构的影响;重组于云母表面人工磷脂膜上的AChE G4结构与底物抑制作用;丁酰胆碱(BCh)对重组于云母表面人工磷脂膜上的AChE G4结构的影响。方法:冰浴超声法制备磷脂膜;囊泡融合技术将AChE重组到以云母为支撑的磷脂膜上;透射电镜(TEM)和原子力显微技术(AFM)对比观察AChE的多分子型;快速蛋白液相色谱法(FPLC)法对AChE G4和AChE G1进行分离、纯化;AFM观察ACh或propidium (PAS抑制剂) -ACh等作用前后AChE G4的亚分子结构动态改变,以及BCh对重组在人工磷脂膜上的AChE G4亚分子结构的影响。结果:制备的以云母为支撑的人工磷脂膜均匀而平坦,融合后缺陷较少,测得形成磷脂膜的厚度是( 2.3±0.3 nm, n=30 ),这是单层磷脂膜的厚度。相同浓度的大豆卵磷脂成膜性不如蛋黄卵磷脂,膜中形成的缺陷较多,粗糙度明显高于蛋黄卵磷脂形成的磷脂膜的粗糙度。在AFM的相位像中,磷脂膜的边界呈高亮显示,可看到与高度像上对应的酶蛋白均匀地镶嵌在磷脂膜上,酶蛋白比磷脂膜暗,表示酶蛋白的粘性比磷脂膜高,弹性比磷脂膜大。单个重组在以云母为支撑人工磷脂膜上的AChE G4(蛋白处于近生理条件下,非晶体状态)颗粒是边界清晰、中间突出的椭圆形球体。构成一个AChE G4颗粒的亚基之间排列紧密,未见亚基构成,蛋白平均大小为[(89±7) nm×(68±9) nm×(6±3) nm,长×宽×高,n=100]。单个重组在以云母为支撑人工磷脂膜上的AChE G1的大小为[(18±5)nm×(15±4)nm×(4.02±0.67)nm,长×宽×高, n=100]。低剂量7μM S-ACh对重组在以云母为支撑人工磷脂膜上的AChE G4亚分子结构最明显的影响是亚基排列松散,粗糙度值明显增大,亚基之间形成一个无阻碍空间,即在四聚体的中间形成一个中央通道,这与Χ-射线晶体学和分子动力学研究结果相一致,中央通道依次经小→大→小→侧门变化,单个AChE G4颗粒平均大小为[ (104±7) nm×(91±5) nm×(8±2) nm,长×宽×高, n=100],无阻碍空间大小为[(60±5) nm×(51±9) nm,长×宽,n=30],侧门口宽(52±5)nm,纵深(32±3)nm,n=10;而在PAS抑制剂存在下,ACh不能引起AChE G4亚分子结构发生变化。在50μM S- ACh作用下,AChE G4的结构出现了明显不同于7μM S-ACh引起的酶结构的变化,可看到形成了多个大小不一的环形蛋白结构,直径为350～400nm,环形结构彼此之间有连接;也有没有发生明显变化的蛋白,直径在150～170nm,没有发生变化的蛋白通常是孤立的。在125μM S-ACh的作用下,酶仍然均匀分布在磷脂膜上,但酶的大小发生了明显的变化,平均大小为[(16±2)nm×(15±1)nm×(1±0.2)nm,长×宽×高, n=100],酶的高度明显降低。与7μM S-BCh反应后,构成AChE G4的四个亚基排列也会出现松散态,酶的大小为[ (100±5) nm×(87±6) nm×(7±3) nm,长×宽×高,n=100],亚基之间形成了一个无阻碍空间,空间的大小比S-ACh作用后的要小,为[(38±4)nm×(35±2)nm,长×宽,n=30]。结论:利用AFM可观察到磷脂膜的微观形成过程为脂质体囊泡吸附→脂质体囊泡之间发生粘着→脂双层之间形成接触点→磷脂囊泡破裂→形成磷脂分子层→磷脂分子层间流动进行融合;通过AFM相位像中磷脂膜和蛋白的粘弹性差别信息以及高亮显示的膜边界可以证明囊泡融合法成功地将AChE组装到了人工磷脂膜上。根据在低剂量7μM ACh作用下,AChE G4亚分子结构的变化,我们提出了AChE实现其高效性的基本原理:AChE G4快速水解ACh的可能关键步骤为:①在正常状态下,处于紧密态的AChE G4只有两个PAS是相对暴露的,在ACh的进攻下,AChE产生强的静电场能吸引阳离子底物ACh与暴露的PAS结合,②单个亚基ACG开放,ACh进入狭隙底部,③底物被水解,④导致亚基之间瞬间斥力增大,四聚体之间形成中央通道,同时亚基之间通过变构使四个PAS均更加暴露,⑤与单个亚基活性位点结合的产物胆碱和乙酸可经亚基“后门”处到达中央通道,⑥酶分子的“侧门”开放,酶分子紧缩,⑦产物离开酶,⑧酶再恢复原态。底物ACh引起AChE G4亚分子结构的变化与其水解底物的高效性是一致的,AFM从形态学上证明了在底物ACh作用下AChE G4分子中间形成中央通道可能控制着酶的周转速度。ACh与PAS的相互作用控制着AChE G4中央通道的产生及单个亚基活性中心狭隙的开放,PAS抑制剂可阻断这种作用。在50μM ACh作用下, AChE为了达到快速水解底物的作用,形成的大的环形结构可能是由2～3个AChE G4形成的开放式通道,多个AChE G4间形成的开放式通道是与其水解底物的高效性相一致的。在125μM ACh作用下, AChE G4会发生底物抑制现象,是由于AChE G4解聚,变成了酶单体,最终导致发生底物抑制现象的是AChE的单体结构。AChE G4与7μM S-BCh作用后构成酶的四个亚基排列也会出现松散态,亚基之间形成了一个无阻碍空间,空间的大小比S-ACh作用后的要小,这可能是AChE水解BCh比水解ACh速度慢的原因之一。
Objective: To explore the possible mechanism of high efficiency of acetylcholinesterase (AChE) hydrolyzing substrates, and study the effects of substrates and inhibitor on the submolecular structures of AChE tetramer (AChE G4) incorporated in a mica-supported artificial phospholipid membrane. Contents:The preparation of artificial phospholipid membrane reconstituted on freshly cleaved mica.The incorporation of AChE in a mica-supported phospholipids membrane.The morphologic study of the molecular isoforms of AChE. The separation and purification of AChE G4 and AChE G1.The effects of ACh and inhibitor on the submolecular structures of AChE G4 incorporated in a mica-supported artificial phospholipid membrane. The submolecular structures of AChE G4 incorporated in a mica-supported artificial phospholipid membrane and the substrate inhibition of AChE. The effects of BCh on the submolecular structures of AChE G4 incorporated in a mica-supported artificial phospholipid membrane. Methods: Ice-bath ultrasound was used to prepare phospholipid membrane. Ves-fusion technique was applied to the reconstitution of AChE G4 in the phospholipid membrane on mica.The phase imaging of AFM-Tapping mode was applied to verify whether AChEs were reconstituted in the phospholipid membrane or not. The molecular isoforms of AChE were observed with atomic force microscope (AFM) and transmission electron microscopy (TEM). FPLC was used to separate and purify AChE G4 and AChE G1. The changes of submolecular structure of AChE G4 incorporated in a mica-supported artificial lipid layer were imaged with AFM before and after reacted with substrate acetylcholine (ACh) with or without the existence of propidium (PAS inhibitor).The changes of submolecular structure of AChE G4 were also imaged with AFM before and after reacted with 7μM butyrocholine (BCh). Results: The mica-supported artificial phospholipid membrane was even, flat and there was not obvious defects, and the height of phospholipid membrane was (2.3±0.3) nm, which was the thickness of phospholipid monolayer. The ability of forming membrane of soybean lecithin is inferior to ovolecithin at the same concentration, and soybean lecithin had more big sizes of defects and higher roughness than ovolecithin. The borders of phospholipid membrane displayed by AFM phase imaging were highlighted, and enzyme particles were seen clearly to be inlayed uniformly in the phospholipid membrane compared with those of AFM height imaging, and enzyme particles were darker than phospholipid membrane, demonstrating the elasticity and viscosity of enzymes were bigger than those of phospholipid membrane. Before reacted with substrates, single AChE G4 particle was ellipsoid in shape, and had smooth surface with a central projection and clear border and the four subunits of single enzyme particle were arranged tightly, no separated subunits being seen, with a mean size of (89±7) nm long, (68±9) nm wide and (6±3) nm high. The size of single AChE G1 particle was(18±5)nm long×( 15±4)nm wide×(4.02±0.67)nm high. After reacted with 7μM S-ACh, loose arrangement of subunits of G4 AChE was seen, with the mean size of (104±7) nm long, (91±5) nm wide and (8±2) nm high, and there was an apparent free space in the middle of the four subunits of the AChE G4, which was consistent with the results of theΧ–ray diffraction crystallography and molecular dynamics study. The apparent free space was the central path of AChE G4, changing from small to big to small to lateral door appearance, with the mean size of (60±5)nm long and (51±9)nm wide. The mean size of lateral door was (52±5) nm wide and (32±3) nm deep. In the presence of PAS inhibitor, ACh couldn’t cause topological structure changes of AChE G4.The changes of submolecular structure AChE G4 reacted with 50μM S-ACh were obviously different from those reacted with 7μM S-ACh, forming some ring-like structures among some proteins.The diameters of these structures were from 350nm to 400nm, and there were linkages between them. At the same time, there were some separated proteins with the diameter of 150nm to 170nm, not forming analogous structures like rings among proteins. The sizes of AChE G4 reacted with 125μM S-ACh were apparently smaller than those reacted with 7μM and 50μM S-ACh, with the mean size of (16±2) nm long, (15±1) nm wide and (1±0.2) nm high. The enzymes after reacted with 7μM S-BCh remained to be distributed on the artificial phospholipid membranes, with mean sizes of (100±5) nm long, (87±6)nm wide and (7±3)nm high, and there was a (38±4)nm long and (35±2)nm wide apparent free space among the subunits of the enzyme. Conclusion: The microscopic forming procedures of phospholipid membrane could be observed with AFM, which is from the adsorption of liposomes to the adherence to form the point of touching between the phospholipid layers to the ruptures of liposomes to form big phospholipid layer and to the fusion among big phospholipid layers. AChE could be verified to be reconstituted successfully in the phospholipid membrane by the difference of elasticity and viscosity between proteins and membranes and the highlight borders of membranes. According to the changes of submolecular structure AChE G4 after reacted with 7μM S-ACh, we raised the possible mechanism of high efficiency AChE of hydrolyzing substrates and the possible key procures as follows:①there are only two PASs of tightly wild AChE G4 exposed, and cationic substrate AChs are attracted by the strong electrostatic field of AChE to combine with exposed PASs at the presence of ACh,②ACG of monomer of AChE G4 is open, and AChs enter the bottom of ACG,③substrates are hydrolyzed,④the repulsion among subunits of AChE G4 are increased flashily, forming a central path among them, and the four PASs are even more exposed by the allosterism of subunits,⑤the products choline and acetic acid combined with the active site of monomer of AChE G4 arrive at the central path via the“back door”of monomer,⑥“lateral door”of the enzyme is open, and enzyme tightens further,⑦products leave enzyme,⑧enzyme recovers to the normal state. The changes of the submolecular structure of AChE G4 are adapted to the high efficiency of AChE hydrolyzing substrates. AFM verified the central path might govern the turnover of the enzyme morphologically and the interactions between PAS and ACh might gate the creation of central path and the open of ACG in monomer; and the combination of ACh with PAS is conducive to the open of ACG while PAS inhibitor can inhibit this action. Some AChE G4s after reacted with 50μM S-ACh form big ring-like structures might be an open path among 2～3 AChE G4 to achieve the rapid hydrolysis of ACh, which is in accordance with the high efficiency of AChE. The phenomenon of inhibition of substrates of AChE G4 after reacted with 125μM S- ACh is brought about from the depolymerization of AChE G4, and the result of the inhibition of substrates is originated from monomer of AChE. The arrangement of four subnnits of AChE G4 after reacted with 7μM S-BCh is the same loose as those after reacted with 7μM S-ACh, and there is a smaller apparent free space than those after reacted with the same concentrational ACh, which is the possible reason of slower speed of AChE hydrolysing BCh than ACh. Resolution at inframolecular level is favourable to provide substantial information on the relative orientations of the subunits within the polymer of enzyme under the effect of substrates with or without the existence of inhibitor.
Objective: To explore the possible mechanism of high efficiency of acetylcholinesterase (AChE) hydrolyzing substrates, and study the effects of substrates and inhibitor on the submolecular structures of AChE tetramer (AChE G4) incorporated in a mica-supported artificial phospholipid membrane. Contents:The preparation of artificial phospholipid membrane reconstituted on freshly cleaved mica.The incorporation of AChE in a mica-supported phospholipids membrane.The morphologic study of the molecular isoforms of AChE. The separation and purification of AChE G4 and AChE G1.The effects of ACh and inhibitor on the submolecular structures of AChE G4 incorporated in a mica-supported artificial phospholipid membrane. The submolecular structures of AChE G4 incorporated in a mica-supported artificial phospholipid membrane and the substrate inhibition of AChE. The effects of BCh on the submolecular structures of AChE G4 incorporated in a mica-supported artificial phospholipid membrane. Methods: Ice-bath ultrasound was used to prepare phospholipid membrane. Ves-fusion technique was applied to the reconstitution of AChE G4 in the phospholipid membrane on mica.The phase imaging of AFM-Tapping mode was applied to verify whether AChEs were reconstituted in the phospholipid membrane or not. The molecular isoforms of AChE were observed with atomic force microscope (AFM) and transmission electron microscopy (TEM). FPLC was used to separate and purify AChE G4 and AChE G1. The changes of submolecular structure of AChE G4 incorporated in a mica-supported artificial lipid layer were imaged with AFM before and after reacted with substrate acetylcholine (ACh) with or without the existence of propidium (PAS inhibitor).The changes of submolecular structure of AChE G4 were also imaged with AFM before and after reacted with 7μM butyrocholine (BCh). Results: The mica-supported artificial phospholipid membrane was even, flat and there was not obvious defects, and the height of phospholipid membrane was (2.3±0.3) nm, which was the thickness of phospholipid monolayer. The ability of forming membrane of soybean lecithin is inferior to ovolecithin at the same concentration, and soybean lecithin had more big sizes of defects and higher roughness than ovolecithin. The borders of phospholipid membrane displayed by AFM phase imaging were highlighted, and enzyme particles were seen clearly to be inlayed uniformly in the phospholipid membrane compared with those of AFM height imaging, and enzyme particles were darker than phospholipid membrane, demonstrating the elasticity and viscosity of enzymes were bigger than those of phospholipid membrane. Before reacted with substrates, single AChE G4 particle was ellipsoid in shape, and had smooth surface with a central projection and clear border and the four subunits of single enzyme particle were arranged tightly, no separated subunits being seen, with a mean size of (89±7) nm long, (68±9) nm wide and (6±3) nm high. The size of single AChE G1 particle was(18±5)nm long×( 15±4)nm wide×(4.02±0.67)nm high. After reacted with 7μM S-ACh, loose arrangement of subunits of G4 AChE was seen, with the mean size of (104±7) nm long, (91±5) nm wide and (8±2) nm high, and there was an apparent free space in the middle of the four subunits of the AChE G4, which was consistent with the results of theΧ–ray diffraction crystallography and molecular dynamics study. The apparent free space was the central path of AChE G4, changing from small to big to small to lateral door appearance, with the mean size of (60±5)nm long and (51±9)nm wide. The mean size of lateral door was (52±5) nm wide and (32±3) nm deep. In the presence of PAS inhibitor, ACh couldn’t cause topological structure changes of AChE G4.The changes of submolecular structure AChE G4 reacted with 50μM S-ACh were obviously different from those reacted with 7μM S-ACh, forming some ring-like structures among some proteins.The diameters of these structures were from 350nm to 400nm, and there were linkages between them. At the same time, there were some separated proteins with the diameter of 150nm to 170nm, not forming analogous structures like rings among proteins. The sizes of AChE G4 reacted with 125μM S-ACh were apparently smaller than those reacted with 7μM and 50μM S-ACh, with the mean size of (16±2) nm long, (15±1) nm wide and (1±0.2) nm high. The enzymes after reacted with 7μM S-BCh remained to be distributed on the artificial phospholipid membranes, with mean sizes of (100±5) nm long, (87±6)nm wide and (7±3)nm high, and there was a (38±4)nm long and (35±2)nm wide apparent free space among the subunits of the enzyme. Conclusion: The microscopic forming procedures of phospholipid membrane could be observed with AFM, which is from the adsorption of liposomes to the adherence to form the point of touching between the phospholipid layers to the ruptures of liposomes to form big phospholipid layer and to the fusion among big phospholipid layers. AChE could be verified to be reconstituted successfully in the phospholipid membrane by the difference of elasticity and viscosity between proteins and membranes and the highlight borders of membranes. According to the changes of submolecular structure AChE G4 after reacted with 7μM S-ACh, we raised the possible mechanism of high efficiency AChE of hydrolyzing substrates and the possible key procures as follows:①there are only two PASs of tightly wild AChE G4 exposed, and cationic substrate AChs are attracted by the strong electrostatic field of AChE to combine with exposed PASs at the presence of ACh,②ACG of monomer of AChE G4 is open, and AChs enter the bottom of ACG,③substrates are hydrolyzed,④the repulsion among subunits of AChE G4 are increased flashily, forming a central path among them, and the four PASs are even more exposed by the allosterism of subunits,⑤the products choline and acetic acid combined with the active site of monomer of AChE G4 arrive at the central path via the“back door”of monomer,⑥“lateral door”of the enzyme is open, and enzyme tightens further,⑦products leave enzyme,⑧enzyme recovers to the normal state. The changes of the submolecular structure of AChE G4 are adapted to the high efficiency of AChE hydrolyzing substrates. AFM verified the central path might govern the turnover of the enzyme morphologically and the interactions between PAS and ACh might gate the creation of central path and the open of ACG in monomer; and the combination of ACh with PAS is conducive to the open of ACG while PAS inhibitor can inhibit this action. Some AChE G4s after reacted with 50μM S-ACh form big ring-like structures might be an open path among 2～3 AChE G4 to achieve the rapid hydrolysis of ACh, which is in accordance with the high efficiency of AChE. The phenomenon of inhibition of substrates of AChE G4 after reacted with 125μM S- ACh is brought about from the depolymerization of AChE G4, and the result of the inhibition of substrates is originated from monomer of AChE. The arrangement of four subnnits of AChE G4 after reacted with 7μM S-BCh is the same loose as those after reacted with 7μM S-ACh, and there is a smaller apparent free space than those after reacted with the same concentrational ACh, which is the possible reason of slower speed of AChE hydrolysing BCh than ACh. Resolution at inframolecular level is favourable to provide substantial information on the relative orientations of the subunits within the polymer of enzyme under the effect of substrates with or without the existence of inhibitor.
引文
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