乙酰胆碱酯酶活性中心狭隙开口的原子力显微观察
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摘要
乙酰胆碱酯酶(AChE)是胆碱能神经系统重要的酶，AChE的作用机理一直是生命科学的重要课题。原子力显微技术(AFM)作为一门新发展起来的技术，提供了人们认识酶的结构与功能的又一新的窗口。本实验利用原子力显微镜(AFM)观察研究了重组于生物膜上的正常乙酰胆碱酯酶分子和与底物乙酰胆碱(ACh)反应后酶蛋白的分子结构，进一步从乙酰胆碱酯酶结构变化上研究其作用机制。主要结果：利用AFM成像可以观察到正常AChE表面平滑，边界清晰，中间突出，略呈椭圆球体，三维显示表现为火山状；与乙酰胆碱作用后，蛋白颗粒直径增大，并且出现成环状态改变。酶蛋白表面变得粗糙，边界模糊，形态也变得不甚规则，蛋白颗粒中心出现近圆形孔洞。三维显示表现为火山口状。初步认为这种近圆形孔洞是AChE活性中心狭隙的开口。AChE活性中心狭隙开口具有两种状态：即关闭和开放状态或狭窄和增宽状态。在未与ACh反应时，它处于关闭或狭窄状态；在突触末梢释放ACh，作用于AChE后，AChE活性中心狭隙开放或者增宽，以利于大量ACh进出AChE的活性中心，满足AChE快速水解的需要。由于AChE的水解速度几乎是由ACh的扩散速度所控制的，因此狭隙的关闭与开放或狭窄与增宽对于AChE的水解速度是极其重要的一个先决条件。可以认为AChE狭隙的增宽或开放是AChE快速水解ACh的结构基础之一，应作进一步的研究。
Acetylcholinesterase (AChE) is an essential enzyme in the cholinergic nervous system. Its principal biological role is to terminate the synaptic signal by catalyzing the hydrolysis reaction of the neurotransmitter, acetylcholine (ACh). Understanding the pharmacological mechanism of AChE to hydrolyze ACh rapidly is of great importance in life science. The newly developed atomic force microscopy (AFM) has opened a new window to the microworld of cells, subcellular structures and biomolecules. In the present study, we observed the structure of the native AChE reconstituted in phospholipid membrane and the structure of AChE reacted with ACh by AFM respectively, in order to make clear the relationship of the rapid hydrolyzing mechanism of AChE to its ultrastructure. The results showed that the appearance of the native AChE was elliptic and smooth, the average long-diameter was94nm, the average short-diameter was 53.01nm and the average height was 3.359nm.The appearance of AChE after reacted with ACh was rough and
the shape became irregular. Most particles had holes in the middle. These results demonstrated that AFM can be used to study the structure of enzyme and the reaction of enzyme with its substrate. The phenomenon that the particles of AChE had holes after reacted with ACh suggests that the active site gorge of AChE probably has two states: close-open or narrow-broad. The gorge is closed or narrow when there are no ACh and is open or wide when there is ACh released from the presynaptic ending. The substrate ACh has an active counter-reaction on the ultrastructure of AChE. It makes the active gorge open or widened to fascilitates more ACh to enter the active site through the active gorge to meet the needs of the rapid hydrolyzing function of the enzyme.
Acetylcholinesterase (AChE) is an essential enzyme in the cholinergic nervous system. Its principal biological role is to terminate the synaptic signal by catalyzing the hydrolysis reaction of the neurotransmitter, acetylcholine (ACh). Understanding the pharmacological mechanism of AChE to hydrolyze ACh rapidly is of great importance in life science. The newly developed atomic force microscopy (AFM) has opened a new window to the microworld of cells, subcellular structures and biomolecules. In the present study, we observed the structure of the native AChE reconstituted in phospholipid membrane and the structure of AChE reacted with ACh by AFM respectively, in order to make clear the relationship of the rapid hydrolyzing mechanism of AChE to its ultrastructure. The results showed that the appearance of the native AChE was elliptic and smooth, the average long-diameter was94nm, the average short-diameter was 53.01nm and the average height was 3.359nm.The appearance of AChE after reacted with ACh was rough and
the shape became irregular. Most particles had holes in the middle. These results demonstrated that AFM can be used to study the structure of enzyme and the reaction of enzyme with its substrate. The phenomenon that the particles of AChE had holes after reacted with ACh suggests that the active site gorge of AChE probably has two states: close-open or narrow-broad. The gorge is closed or narrow when there are no ACh and is open or wide when there is ACh released from the presynaptic ending. The substrate ACh has an active counter-reaction on the ultrastructure of AChE. It makes the active gorge open or widened to fascilitates more ACh to enter the active site through the active gorge to meet the needs of the rapid hydrolyzing function of the enzyme.
引文
1、林煜，陈俊抛等．大鼠海马注射β淀粉样蛋白建立记忆障碍模型的研究，中华精神科杂志，2000(4)：222-225
2、盛树立，裴进京．老年性痴呆的临床和分子生物学基础．科学技术文献出版社，1998，10
3、Rump, S.; Kowalczyk, M.; Gidynska, T.; Galecka, E.; Faff, J. Lack of AChE reactivation and effects of ACh synthesis inhibition in protection against soman toxicity. Toxicology Letters, 1998(95), 151
4、Sussman JL, Harel M, Frolow F, et al. Atomic structure of AChE from Torpedo californica: a prototypic acetylcholine-binding protein. Science, 1991,253:872-879
5、Binnig G, Quate CF, Gerber C. Atomic force microscope. Phys Rev Lett, 1986, 56(9):930
6、Mou, J., Czajkowsky, D.M., Zhang, Y. and Shao, Z. (1995) FEBS Lett., 371,279-282
7、Hansma, H. G., Bezanilla, M., Zenhausern, F., Adrian, M. and Sinsheimer, R.L.(1993) Nucleic Acids Res., 21,505-512
8、Lyubchenko,Y.L. and Shlyakhtenko, L.S.(1997) Proc.Natl.Acad.Sci.USA, 94: 496-501
9、Osmulski PA and Gacynzynska M. Atomic force microscopy reveals two conformations of the 20S proteasome from Fission yeast[J]. J Bili Chem, 2000 275(18):13171
10、Konstantin Balashev, Torben R. Jensen, Kristian Kjaer. Novel methods for studying lipids and their mutual interaction at interfaces. Part Ⅰ. Atomic force microscopy. Biochimie 2001(83): 387-397
11、刘冰，原子力显微镜及其在生物物理学，2003
12、焦克芳，曲红，李松．胆碱酯酶与小分子底物相互作用的计算机模拟研究．军事医学科学院院刊，1998，22(3)：195-198，202单分子研究中的应用．《分子生物
13、Y Dudai, I Silman, N Kalderon, and S Blumberg. Purification by affinity
chromatography of acetylcholinesterase from electric organ tissue of the electric eel subsequent to tryptic treatment. Biochim Biophys Acta, Apr 1972; 268(1): 138-57.
14、孙曼霁，高天栋，邢志勇等，中国丁氏双鳍电鳐乙酰胆碱酯酶的制备亲和层析，生物化学杂志，1985，1：47
15、New, R.R.C. (1990) in Liposomes a practical approach (New, R.R.C., ed.), pp33-103, Information Press, Oxford.
16、赵南明，周海梦，《生物物理学》，高等教育出版社，施普林格出版社
17、Shao, Z., Mou, J., Czajkowsky, D.M., Yang, J. and Yuan, J.-Y. Biological atomic force microscopy: what is achieved and what is needed. (1996) Adv. Phys. 45, 1-86
18、Blodgett,K.B. Films built by depositing successive monomolecular layers on a solid surface. J. Am. Chem. Soc. 57(1935), 1007-1022.
19、J Mou, J Yang, and Z Shao. Tris (hydroxymethyl) aminomethane (C4H11NO3) induced a ripple phase in supported unilamellar phospholipid bilayers. Biochemistry, Apr 1994; 33(15): 4439-43.
20、余和芬，陈珈，脂质体重组和脂蛋白体在植物生物膜研究中的应用，植物学通报，2000，17(2)：150-154
21、Y Fang and J Yang. The growth of bilayer defects and the induction of interdigitated domains in the lipid-loss process of supported phospholipid bilayers. Biochim Biophys Acta, Mar 1997; 1324(2): 309-19.
22、Leila Dziri, Salah Boussaad, Surface topography of acetylcholinesterase in Langmuir and Langmuir-Blodgett films. J.Phys.Chem.B 1997, 101,6741-6748
23、Kasahara M, Hinkle PC, 1977. Reconstitution and Purification of the D-Glucose Transporter from Human Erythrocytes. J Biol Chem, 252:7384-7392
24、Kagawa Y, Racher E, 1971. Partial Resolution of the Enzymes Catalyzing Oxidative Phosphorylation. J Biol Chem, 246:5477-5484
25、BanNs J, Ratajczak R, Lüttge U, 1994. Solubilization and functional reconstitution of the Tonoplast H~+-ATPase from Citrus in liposomes. J Plant physiol, 114:74-79
26、Kasamo K, Yamanishi H, 1991. Functional Reconstitution of Plasma Membrane
H~+-ATPase from Mung Bean (Vigna radiata L.) hypocotyls in Liposomes Prepared with Various Molecular Species of Phospholipids. Plant Cell Physiol, 32(8): 1219-1225
27、Yadin dudai, Max herzberg, Israel silman. Molecular structure of acetylcholine-sterase from Electric organ tissue of the Electric eel. Proc. Nat. Acad. Sci. USA 1973(70), 2473-2476
28、张英鸽，王琛，赵德禄等．乙酰胆碱酯酶分子的原子力显微成像，电子显微学报，1999(18)：94-98．
29、Kunio Takeyasu, Hiroshi Omote, Saju Nettikadan, Molecular imaging of Escherichia coli F_0F_1-ATPase in reconstituted membranes using atomic force microscopy. FEBS Letters 1996(392): 110-113.
30、Moral Naranjo MT, Cabezas Herrera J, Vidal CJ. Molecular forms of acetyl- and butyryl- cholinesterase in normal and dystrophic mouse brain [J]. J Neurosci Res, 1996, 43(3): 224.
31、Pawel A, Osmulski and Maria Gaczynska. Atomic force microscopy reveals two conformations of the 20S proteasome from Fission Yeast. J Biol Chem, 2000, 275(18): 13171-13174
2、盛树立，裴进京．老年性痴呆的临床和分子生物学基础．科学技术文献出版社，1998，10
3、Rump, S.; Kowalczyk, M.; Gidynska, T.; Galecka, E.; Faff, J. Lack of AChE reactivation and effects of ACh synthesis inhibition in protection against soman toxicity. Toxicology Letters, 1998(95), 151
4、Sussman JL, Harel M, Frolow F, et al. Atomic structure of AChE from Torpedo californica: a prototypic acetylcholine-binding protein. Science, 1991,253:872-879
5、Binnig G, Quate CF, Gerber C. Atomic force microscope. Phys Rev Lett, 1986, 56(9):930
6、Mou, J., Czajkowsky, D.M., Zhang, Y. and Shao, Z. (1995) FEBS Lett., 371,279-282
7、Hansma, H. G., Bezanilla, M., Zenhausern, F., Adrian, M. and Sinsheimer, R.L.(1993) Nucleic Acids Res., 21,505-512
8、Lyubchenko,Y.L. and Shlyakhtenko, L.S.(1997) Proc.Natl.Acad.Sci.USA, 94: 496-501
9、Osmulski PA and Gacynzynska M. Atomic force microscopy reveals two conformations of the 20S proteasome from Fission yeast[J]. J Bili Chem, 2000 275(18):13171
10、Konstantin Balashev, Torben R. Jensen, Kristian Kjaer. Novel methods for studying lipids and their mutual interaction at interfaces. Part Ⅰ. Atomic force microscopy. Biochimie 2001(83): 387-397
11、刘冰，原子力显微镜及其在生物物理学，2003
12、焦克芳，曲红，李松．胆碱酯酶与小分子底物相互作用的计算机模拟研究．军事医学科学院院刊，1998，22(3)：195-198，202单分子研究中的应用．《分子生物
13、Y Dudai, I Silman, N Kalderon, and S Blumberg. Purification by affinity
chromatography of acetylcholinesterase from electric organ tissue of the electric eel subsequent to tryptic treatment. Biochim Biophys Acta, Apr 1972; 268(1): 138-57.
14、孙曼霁，高天栋，邢志勇等，中国丁氏双鳍电鳐乙酰胆碱酯酶的制备亲和层析，生物化学杂志，1985，1：47
15、New, R.R.C. (1990) in Liposomes a practical approach (New, R.R.C., ed.), pp33-103, Information Press, Oxford.
16、赵南明，周海梦，《生物物理学》，高等教育出版社，施普林格出版社
17、Shao, Z., Mou, J., Czajkowsky, D.M., Yang, J. and Yuan, J.-Y. Biological atomic force microscopy: what is achieved and what is needed. (1996) Adv. Phys. 45, 1-86
18、Blodgett,K.B. Films built by depositing successive monomolecular layers on a solid surface. J. Am. Chem. Soc. 57(1935), 1007-1022.
19、J Mou, J Yang, and Z Shao. Tris (hydroxymethyl) aminomethane (C4H11NO3) induced a ripple phase in supported unilamellar phospholipid bilayers. Biochemistry, Apr 1994; 33(15): 4439-43.
20、余和芬，陈珈，脂质体重组和脂蛋白体在植物生物膜研究中的应用，植物学通报，2000，17(2)：150-154
21、Y Fang and J Yang. The growth of bilayer defects and the induction of interdigitated domains in the lipid-loss process of supported phospholipid bilayers. Biochim Biophys Acta, Mar 1997; 1324(2): 309-19.
22、Leila Dziri, Salah Boussaad, Surface topography of acetylcholinesterase in Langmuir and Langmuir-Blodgett films. J.Phys.Chem.B 1997, 101,6741-6748
23、Kasahara M, Hinkle PC, 1977. Reconstitution and Purification of the D-Glucose Transporter from Human Erythrocytes. J Biol Chem, 252:7384-7392
24、Kagawa Y, Racher E, 1971. Partial Resolution of the Enzymes Catalyzing Oxidative Phosphorylation. J Biol Chem, 246:5477-5484
25、BanNs J, Ratajczak R, Lüttge U, 1994. Solubilization and functional reconstitution of the Tonoplast H~+-ATPase from Citrus in liposomes. J Plant physiol, 114:74-79
26、Kasamo K, Yamanishi H, 1991. Functional Reconstitution of Plasma Membrane
H~+-ATPase from Mung Bean (Vigna radiata L.) hypocotyls in Liposomes Prepared with Various Molecular Species of Phospholipids. Plant Cell Physiol, 32(8): 1219-1225
27、Yadin dudai, Max herzberg, Israel silman. Molecular structure of acetylcholine-sterase from Electric organ tissue of the Electric eel. Proc. Nat. Acad. Sci. USA 1973(70), 2473-2476
28、张英鸽，王琛，赵德禄等．乙酰胆碱酯酶分子的原子力显微成像，电子显微学报，1999(18)：94-98．
29、Kunio Takeyasu, Hiroshi Omote, Saju Nettikadan, Molecular imaging of Escherichia coli F_0F_1-ATPase in reconstituted membranes using atomic force microscopy. FEBS Letters 1996(392): 110-113.
30、Moral Naranjo MT, Cabezas Herrera J, Vidal CJ. Molecular forms of acetyl- and butyryl- cholinesterase in normal and dystrophic mouse brain [J]. J Neurosci Res, 1996, 43(3): 224.
31、Pawel A, Osmulski and Maria Gaczynska. Atomic force microscopy reveals two conformations of the 20S proteasome from Fission Yeast. J Biol Chem, 2000, 275(18): 13171-13174