Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/16669
標題: 新抑癌素蛋白D79殘基負電荷對引導生色團環化路徑之影響
Effect of D79 negative charge of neocarzinostatin protein on directing cyclization pathway of its chromophore
作者: 鄭靖修
Cheng, Ching-Hsiu
關鍵字: neocarzinostatin;新抑癌素;enediyne;antitumor;cycloaromatization mechanism;HSQC NMR;protein mutanttion;烯雙炔;環化機制;抗腫瘤;異種核磁共振;蛋白突變
出版社: 化學系所
引用: 1.Ishidan, N., Miyazaki, K., Kumagai, K., Rikimaru, M. Neocarzinostatin, an antitumor antibiotic of high molecular weight. Isolation, physiochemical properties and biological activites. Journal of Antibiotics (Tokyo), 1965, 18, 68-76 2.Kuromizo, K., Abe, O., Maeda, H. Location of the disulfide bonds in the antitumor protein neocarzinostatin. Archives of Biochemistry and Biophysics, 1991, 286 (2), 569-573 3.Kim, K. H., Kwon, B. H., Myers, A. G., Rees, D. C. Crystal structure of neocarzinostatin, an antitumor protein-chromophore complex. Science, 1993, 262 (5136), 1042-1046 4.Albers-Schonberg, G., Dewey, R. S., Hensens, O. D., Liesch, J. M., Napier, M. A., Goldberg, I. H. Neocarzinostatin: chemical characteriza-tion and partial structure of the non-protein chromophore. Biochemical and Biophysical Research Communications, 1980, 95, 1351-1356 5.Edo, K., Mizugaki, M., Koide, Y., Seto, H., Furihata, K., Otake, N., Ishida, N. 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Biochemistry, 1990, 29, 8401 39.Adjadj, E., Mispetlter, J., Quiniou, E., Dimicoli, J. L., Favaudon, V., Lhoste, J. M. Protein NMR studies of apo-neocarzinostatin from Strep-tomyces carzinostaticus. Sequence-specfic assignment and secondary structure. Biochemistry, 1990, 29, 8401 40.Gao, X. L., Burkhart, W. Two- and three-dimernsional protein NMR studies of apo-neocarzinostatin. Biochemistry, 1991, 30, 7730 41.Napier, M. A., Holmquist, B., Strydom, D. J., Goldberg, I. H. Neocarzi-nostatin: spectral characterization and separation of a non-protein chromophore. Biochemical and Biophysical Research Communications, 1979, 89, 635-642 42.Kappen, L. S., Goldberg, I. H. Mechanism of the effect of organic sol-vents and other protein denaturants of neocarzinostatin activity. Biochemistry, 1979, 18 (25), 5647-5653 43.Kappen, L. S., Goldberg, I. H. Stabilization of neocarzinostatin non-protein chromophore activity by interaction with apoprotein and with HeLa cells. 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摘要: 
新抑癌素是一種強力的天然抗腫瘤抗生素,由蛋白與生色團所組成,生色團具有烯雙炔的結構,能夠斷裂DNA而殺死細胞。新抑癌素生色團在沒有蛋白保護下,可以經由硫醇的活化,將烯雙炔環化,產生高活性的自由基,抓取DNA或溶劑的氫原子後,形成穩定的產物一。近年來,對於該機制已有不少研究文獻發表,不過已知的新抑癌素生色團的環化路徑並不止此,當生色團受到蛋白保護時,環化路徑受到蛋白環境的影響而改變,環化形成的主產物由產物一變為產物二,對這種蛋白內進行的失去藥物活性的環化機制,探究並不多,蛋白為何擁有引導環化路徑的能力,至今尚沒有完整的探索。
本實驗室學長偶然間發現,移除新抑癌素蛋白D79殘基的負電荷後,蛋白內環化產物一生成比例顯著上升,於是我們懷疑D79殘基的負電荷能引導生色團環化成產物二。我們利用蛋白工程技術生產數種特性不同的突變蛋白,包括D79殘基的單點突變蛋白,也包括D79以及附近殘基的雙突變蛋白。突變蛋白經過純化後,我們用液相層析、質譜、及紫外光譜等,驗證了突變蛋白分子的正確性及純度。用乙醯化反應驗證了突變蛋白的二個雙硫鍵完整結合。用圓二色光譜驗證了突變蛋白仍保留原有的二、三級結構。用熱變性的實驗測量比較了這些突變蛋白的結構穩定性。我們又選取了二種突變蛋白,生產並純化15N標記的同位素突變蛋白,用二維的核磁共振實驗檢驗蛋白的各個殘基位置,驗證了突變蛋白仍然保有結合生色團的穴口結構。最後我們將萃取的天然新抑癌素生色團與突變蛋白重新結合,置入硫醇,讓生色團在這些突變蛋白內部產生烯雙炔的環化反應,之後用高效能液相層析分析生色團環化後的產物,從產物一及產物二的生成比例變化,我們嘗試探究D79殘基上的負電荷對於新抑癌素生色團環化的影響。
分析環化產物的結果顯現,新抑癌素蛋白D79殘基的負電荷,確實會影響生色團中烯雙炔的環化路徑,我們依據分析的結果並考量電荷效應、新抑癌素生色團化學結構、環張力與混成軌域等要素,提出產生產物二的新環化機理,並藉由簡易的理論計算估計這些環化中間物的生成熱,推敲評估模型的合理性,進一步地比較環化路徑的異同之處,以解釋新抑癌素蛋白如何影響生色團環化成為不同的產物。我們期待提出的產物二的新環化機理,能提供他人以更多角度去看待並解釋烯雙炔分子的環化機制,讓大家能更了解藥物與蛋白載體之間的巧妙天然設計。

Neocarzinostatin is a natural potent antitumor antibiotic chromoprotein. It consists of a carrier protein, aponeocarzinostatin, and a biologically active enediyne chromophore. Without the protein, the enediyne nucleus of neocarzinostatin chromophore is cycloaromatized upon a thiolate attack to form product 1, and the radical precursor of which is responsible for DNA cleavage. Whereas in the holo form of neocarzinostatin, product 1 formation is highly suppressed as the protein diverts the major cycloaromatization path into a distinct one that produces product 2. How the protein directs such a chemical path is an interesting question. We produced recombinant aponeocarzinostatin that is identical to the natural one, and employed the alanine-scanning mutagenesis to probe the influence of residues around binding cleft on directing the enediyne cycloaromatization pathways. We found that mutant D79A significantly abolishes the protein directing power. In this study, we designed and performed several mutations focusing around D79. The circular dichroism spectra of all the mutants show a folded structure that is similar to that of the wild-type aponeocarzinostatin. Two-dimensional nuclear magnetic resonance spectra of purified 15N-labeled D79A and double mutant L77D/D79A demonstrate that most residues around binding site are not perturbed by mutations. The thiol-induced enediyne cycloaromatization reaction was then performed on each chromophore-reconstituted neocarzinostatin mutant, and the yield of product 1 was determined by high pressure liquid chromatographic analyses. Our results clearly show that negative charge of D79 is crucial for directing the chemical cyclization pathway of neocarzinostatin chromophore. Considering ring strain and hybrid orbitals of possible cyclized intermediates, we proposed a mechanism of formation of product 2.
URI: http://hdl.handle.net/11455/16669
其他識別: U0005-1408200811454300
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