AFM观察ACh和PAS抑制剂对AChE四聚体结构的影响

AFM observation of the effects of acetylcholine and PAS inhibitor on the structure of tetrameric acetylcholinesterase

目的:探索乙酰胆碱(ACh)和外周阴离子部位(PAS)在调节乙酰胆碱酯酶四聚体(AChE G4)结构中的作用。方法:冰浴超声法制备磷脂膜,囊泡融合技术将AChE G4重组到以云母为支撑的磷脂膜上,原子力显微技术(AFM)观察与ACh或propidium(PAS抑制剂)-ACh作用前后AChE G4的亚分子结构变化。结果:天然AChE G4的各个亚基排列紧密,未见亚基构成。ACh对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)亚分子结构最明显的影响是亚基排列松散,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,中央通道依次经小→大→小→侧门变化;而在PAS抑制剂存在时,ACh不引起AChE G4亚分子结构变化。结论: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。

The role of acetyleholine and peripheral anionic site （PAS） in adjusting the structure of tetrameric acetylcholinesterase （ACHE G4 ） was investigated. Iced-bath ultrasound was used to prepare phospholipid membrane. Ves-fusion technique was applied in the reconstitution of AChE G4 in the phospholipid membrane on mica. 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 four subunits of single enzyme particle before reacted with substrates were arranged tightly, not seeing separated subunits. Loose arrangement of subunits of G4 AChE is the most obvious effect of ACh. There is an apparent free space in the center of G4 AChE after reacted with ACh. The free space is the central path of AChE G4, changing from small to big to small to lateral door. In the presence of PAS inhibitor, ACh couldn＇ t cause topological structure changes of AChE G4. The changes of the submolecular structure of AChE G4 are adapted to the high efficiency of AChE G4 to hydrolyze 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; the combination of ACh with PAS is conducive to the open of ACG while PAS inhibitor can inhibit this action. Resolution at inframolecular level is favourable to provide substantial information on the relative orientations of the snhunits within the polymer of enzyme under the effect of substrates with or without the existence of PAS inhibitor.