Please use this identifier to cite or link to this item:
標題: 探討人類蘋果酸酶二聚體與四聚體中間介面對結構穩定性之生物物理特性
Biophysical characterization of dimer and tetramer interface interaction in stability of the human malic enzyme.
作者: 蘇繼
Kumar, M.Sujith
關鍵字: malic enzyme
出版社: 生命科學系所
引用: 1. Hsu, R.Y. (1982) Pigeon liver malic enzyme. Mol. Cell Biochem. 43, 3-26. 2. Loeber, G., Infante, A. A., Maurer-Fogy, I., Krystek, E., Dworkin, M. B. (1991) Human NAD(+)-dependent mitochondrial malic enzyme. cDNA cloning, primary structure and expression in Escherichia coli. J. Biol. Chem. 266, 3016-3021. 3. Frenkel, R., (1975) Regulation and physiological functions of malic enzymes. Current Topics in cellular regulation. 9, 157-181. 4. Xu, Y., Bhargava, G., Wu, H., Loeber, G., and Tong, L. (1999) Crystal structure of human mitochondrial NAD(P)+-dependent malic enzyme: a new class of oxidative decarboxylase. Structure 7, 877-889. 5. Loeber, G., Dworkin, M. B., Infante, A., and Ahorn, H. (1994) Characterization of cytosolic malic enzyme in human tumour cells. FEBS Lett. 344, 181-186. 6. Goodridge, A. G., Klautky, S. A., Fantozzi, D. A., Baillie, R. A., Hodnett, D. W., Chen, W., Thurmond, D.C., Xu, G., Roncero, C. (1996) Nutriontional and hormonal regulation of expression of the gene for the malic enzyme. Prog. in Nucleic acid Res. Mol. Biol. 52, 89-122. 7. Frenkel, R. (1975) Regulation and physiological functions of malic enzymes Curr. Top. Cellu. Regul. 9, 157-181. 8. Stryer, L. (1995) Biochemistry, 4th ed, Freeman, New York. 9. Sundqvist, K.E., Heikkila, J., Hassinen, I.E., and Hiltunen, J.K. (1987) Role of NAD+-linked malic enzyme regulatirs of the pool size of tricarboxylic acid cycle intermediates in the perfused rat liver. Biochem. J. 243, 853-857. 10. .Ayala, A., F-Lobato, M., and Machado, A. (1986) Malic enzyme levels are increased by the activation of NADPH- consuming pathways: detoxification processes. FEBS Lett. 202, 102-106. 11. Fukuda, H., Katsurada, A., and Iritani, N. (1990) Effects of aging on transcriptional and post transcriptional regulation of malic enzyme and glucose-6-phospahte dehydragenase in rat liver. Eur. J. Biochem. 188, 517-522. 12. Stumpf, D.A., Parks, J.K., Eguren, L.A., and Haasr, R. (1982) Friedreich ataxia:III. Mitochondrial malic enzyme deficiency. Neurology 32, 221-226. 13. Fernandez, R.J., Civantos, F., Tress, E., Maltese, W.A., and De vivo, D.C. (1986) Normal fibroblast mitochondrial malic enzyme activity in Friedreich's ataxia. Neurology 36, 869-872. 14. Reitzer, L. J., Wice, B. M., and Kennell, D. (19790 evidence that glutamate, not sugar, is the major energy source for cultured HeLa cells. J. Biol. Chem. 254, 2669-2676 15. Mckeehan, W.L. (1982) Glycoloysis, glutaminolysis and cell proliferation. Cell biology International Reports. 6, 635-650. 16. Moreadith, R. W., and Lehninger, A.L. (1984) The pathways of glutamate and glutamine oxidation by tumour cell mitochondria:role of mitochondrial NAD(P)+-dependent malic enzyme. J. Biol. Chem. 259, 6215-6221. 17. Fahien, L.A. and Teller, J.K. (1992) Glutamate-malate metabolism in liver mitochondria. J.Biol.Chem. 267, 10411-10422. 18. Teller, J.K., Fahien, L.A., and Davis, J.W. (1992) Kinetic and regulation of hepatoma mitochondrial NAD(P) malic enzyme. J.Biol.Chem. 267, 10423-10432. 19. Yang, Z., Zhang, H., Hung, H.C., Kuo, C.C., Tsai, L.C., Yuan, H.S., Chou, W.Y., Chang, G.G., and Tong, L. (2002) Structural studies of the pigeon cytosolic NAD(P)+-dependent malic enzyme. Protein Sci. 11, 332-41. 20. Chang, G.G. and Tong, L. (2003) Structure and function of malic enzyme, a new class of oxidative decarboxylases Biochemistry, 42, 12721-12733. 21. Tao, X., Yang, Z., and Tong, L. (2003) Crystal structures of substrate complexes of malic enzyme and insights into catalytic mechanism. Structure. 11, 1141-1150. 22. Yang, Z., Floyd, D. L., Loeber, G., and Tong, L. (2000) Structure of a closed form of human malic enzyme and implications for catalytic mechanism. Nat. Struct. Biol. 7, 251-257. 23. Hsieh, J. Y., Liu, G. Y., and Hung, H. C. (2008) Influential factor contributing to the isoform-specific inhibition by ATP of human mitochondrial NAD(P)+-dependent malic enzyme: functional roles of the nucleotide binding site Lys346. FEBS J. 275, 5383-5392. 24. Karsten, W. E., Pais, J. E., Rao, G. S., Harris, B. G., and Cook, P. F. (2003) Ascaris suum NAD-malic enzyme is activated by L-malate and fumarate binding to separate allosteric sites. Biochemistry 42, 9712-9721. 25. Hung, H. C., Kuo, M. W., Chang, G. G., and Liu, G. Y. (2005) Characterization of the functional role of allosteric site Asp102 in the regulatory mechanism of human mitochondrial NAD(P)+-dependent malic enzyme. Biochem. J. 392, 39-45. 26. Rao, G. S., Coleman, D. E., Karsten, W. E., Cook, P. F., and Harris, B. G. (2003) Crystallographic studies on Ascaris suum NAD-malic enzyme bound to reduced cofactor and identification of an effector site.J. Biol. Chem. 278, 38051-38058. 27. Chang, H.C. and Chang, G.G, (2003) Involvement of single residue tryptophan 548 in the quaternary structural stability of pigeon cytosolic malic enzyme J. Biol. Chem. 278, 23996-24002. 28. Chang, H. C., Chen, L. Y., Lu, Y. H., Li, M. Y., Chen, Y. H., Lin, C. H., and Chang, G. G. (2007) Metal ions stabilize a dimeric molten globule state between the open and closed forms of malic enzyme. Biophys. J. 93, 3977-3988. 29. Chou, W.Y., Huang, S.M., and Chang, G.G. (1997) Functional roles of the N-terminal amino acid residues in Mn(II)-L-Malate binding and subunit interactions of pigeon liver malic enzyme. Protein. Eng. 10, 1205-1211. 30. Chou, W. Y., Liu, M. Y., Huang, S. M., and Chang, G. G. (1996) Involvement of Phe19 in the Mn(2+)-L-malate binding and the subunit interactions of pigeon liver malic enzyme. Biochemistry 35, 9873-9879 31. Timasheff, S.N. (1993) The Control of Protein Stability and Association by Weak Interactions with Water: How Do Solvents Affect These Processes? Annu. Rev. Biophys. Biomol. Struct. 22, 67-97. 32. Murphy, K.P., Freire, E. (1992) Thermodynamics of structural stability and cooperative folding behavior in proteins. Adv. Protein Chem. 43, 313-361. 33. Plapp, B.V. (1995) Site-directed mutagenesis: a tool for studying enzyme catalysis. Methods Enzymol. 249, 91-119. 34. Hsieh, J, Y., Chen, S. H., and Hung, H. C. (2009) Functional Roles of the Tetramer Organization of Malic Enzyme. 27, 18096-18105,
摘要: 蘋果酸酶具有雙二聚體的四級結構,其中二聚體中間介面較四聚體中間介面更為緊密接觸。細胞質蘋果酸酶 (c-NADP-ME) 異構型以穩定四聚體型式存在且不具有協同作用與異位調控的特性。在本篇研究中,我們分析人類細胞質蘋果酸酶之二聚體上-下與左-右型式的結構穩定性。挑選位於二聚體及四聚體中間介面之胺基酸進行於定點突變,分別為第51、90、139、142、568、572號胺基酸並獲得一系列人類細胞質蘋果酸酶之二聚體。我們構築二個單點突變 ( H142A, W572A ) , 三個雙點突變 (H51A/D90A, H51A/D139A, H142A/D568A ),表現於大腸桿菌並進行純化。藉由螢光與原二色光譜儀及 ANS 螢光分析技術,研究蛋白結構在熱變性與尿素誘發變性過程之去摺疊行為。從熱變性的結果得知,二聚體及四聚體中間介面之突變型的熱穩定性具有 ∆Tm ~8-10 °C 的差異。推測由於二聚體中間介面之突變型具有較強的結合力。而在尿素誘發變性過程顯示二聚體中間介面之突變型的第一轉變階段位移至較低的尿素濃度。螢光實驗結果顯示破壞中間介面的結合力進而降低結構穩定性。從 ANS 螢光分析得知,二聚體中間介面之突變型的最大螢光強度出現於低尿素濃度。我們的研究證實人類細胞質蘋果酸酶之結構穩定性不受四聚體中間介面結合力破壞的影響。四聚體中間介面之突變型與野生型呈現相似的結構穩定性。然而,當破壞二聚體中間介面結合力則顯著喪失人類細胞質蘋果酸酶之結構穩定性且二聚體中間介面之突變型的結構穩定性低於野生型。以上結果顯示,相較於四聚體中間介面之結合力,二聚體中間介面之結合力在人類細胞質蘋果酸酶之結構穩定性中扮演著重要角色。
Malic enzyme has a dimer of dimers quaternary structure in which the dimer interface associates more tightly than the tetramer interface. The cytosolic NADP+-dependent malic enzyme (c-NADP-ME) isoform is non-cooperative and non-allosteric and exists as a stable tetramer. In this study, we analyzed the stability of up-and-down dimer and left-and-right dimers of human c-NADP-ME. Site-directed mutagenesis at the dimer and tetramer interfaces was employed to generate a series of dimers of c-NADP-ME. Trp-572, His-142, Asp-568, His-51, Asp-90 and Asp-139 were selected as target sites for mutagenesis, because they are at the subunit interface. W572A, H142A, H142A/D568A, H51A/D90A and H51A/D139A mutant enzymes were constructed, expressed in Escherichia coli, and purified. We studied thermal and urea-induced unfolding using fluorescence and CD spectroscopy, as well as ANS fluorescence. Thus, the difference in thermo stability between tetramer and dimer interface mutants were ∆Tm ~8-10 °C. It is related to the strength of interaction between the dimer interface. Urea-induced unfolding also showed the first transistion shift towards lower urea concentration in dimer interface mutants. The Intrinsic fluorescence studies showed the decrease in stability due to the disruption of interface interaction. The ANS binding studies showed that the maximum intensity observed at lower concentration of urea in dimer interface mutants. Our studies revealed that the enzyme stability of c-NADP-ME is not affected by disruption of the tetramer interface. The analysis of tetramer interface mutants showed similar stability with wild type. However, disruption on the dimer interface showed significant loss of stability of c-NADP-ME and the analysis of the dimer interface mutants showed less stability than the wild type. These results indicate that the interaction between the dimer interface play an important role in the stability of c-NADP-ME than the interactions between the tetramer interface.
其他識別: U0005-0807201014513300
Appears in Collections:生命科學系所



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.