Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/15930
標題: 探討新抑癌素蛋白穴口酸性胺基酸殘基保護生色團的功能
Examining the chromophore-protecting capacity of the acidic residues at the cavity opening site in neocarzinostatin protein.
作者: 黃俊期 論文開放日期:20040816
Huang, Chun-Chi
關鍵字: neocarzinostatin;新抑癌素;protection;charge;thiol;電荷;蛋白保護;硫醇
出版社: 化學系
摘要: 
摘要
新抑癌素 (NCS)是一種天然的強力抗腫瘤抗生素。由一個酸性蛋白 (apoNCS)和生色團 (Chromophore)所組成,其生物活性來自於生色團上所含的烯雙炔環,新抑癌素蛋白對於生色團具有調節的作用並可以保護生色團避免光、熱和其他的親核基的作用。
硫醇,可以使生色團激活或去活化,在此扮演著輔因子的角色,當沒有蛋白的存在時,硫醇會激活生色團,使環化作用,產生自由基,有損傷DNA的能力,在蛋白的存在時,硫醇可以穿越蛋白,使生色團失去活性,但有些硫醇類,例如麩胺基硫―真核細胞中含量最多的硫醇類,卻被蛋白排斥在外,本實驗室曾粗略估算,推測在新抑癌素蛋白穴口上的33、79、99三個酸性胺基酸殘基,對於負電荷的硫醇有排斥的作用,可以保護生色團,避免失去活性,為了進一步證明,我們借用分生的技術,使用定點突變的方法,將新抑癌素蛋白穴口上的33、79、99三個酸性胺基酸殘基,各別置換成中性的丙鞍酸 (D33A、D79A和D99A),為了比較,也將同在穴口76、77和98位置的中性胺基酸殘基,也置換成丙鞍酸 (F76A、L77A和S98A),然後與新抑癌素蛋白比較它們的保護作用。
實驗的結果觀察到,中性胺基酸殘基在經過修飾後,對蛋白保護作用沒有影響,酸性胺基酸殘基修飾成中性的蛋白,確實能夠使帶有負電荷的硫醇穿越蛋白與生色團作用,使新抑癌素蛋白喪失不同程度的保護能力,在初步的結果可以看出這三個位置對於生色團保護的能力分別是33 > 99 > 79,由本實驗室曾做的粗略計算已經知道,這三個位置和生色團第十二號碳的距離分別是79 > 99 > 33,這樣的差距很有可能是導因於這三個胺基酸殘基與生色團第十二號碳的長短距離。然而,實驗的結果有助於進一步了解蛋白對生色團保護作用的機制。

ABSTRACT
Neocarzinostatin (NCS) is the first identified enediyne antitumor antibiotic isolated from Streptomyces carzinostaticus. When the enediyne chromophore is released from apoNCS, thiols can activate the chromophore by attacking at its C12 to generate a biradical intermediate at C2 and C6 by cycloaromatization (Product 1). This biradical can abstract two hydrogens from DNA or other sources. However, if a thiol is allowed by the apoprotein to reach the protein-bound chromophore, another distinct thiol-chromophore adduct is formed (Product 2). This latter type of product is not able to abstract hydrogen from DNA and hence, it does not cleave DNA. Our previous studies stressed the charge mediated selection/rejection of thiols by NCS and envisaged the importance of certain negative amino acid residues around the chromophore binding pocket. To evaluate the hypothesis, in the present study, apoNCS genes with coding potential for mutant proteins were constructed. The mutant proteins were expressed in E. coli, purified and checked by mass spectroscopy for their authenticity. The possible influence of the amino acid substitutions on the structure of apoNCS is assessed by far- and near-UV circular dichroism (CD) spectroscopic analyses. The far-UV CD profiles of mutant apoNCS proteins reveal the preservation of the protein structure similar to that of wild type apoNCS, despite the substitution of aspartic acid residues 33, 79 and 99, phenylalanine residue 76, leucine 77 and serine 98 by alanine residues. However, contrary to the previously reported rejection of negative thiols by wild type NCS, the substitution of aspartic acid residues 33, 79 and 99 by alanine residues allows the negatively charged thiols, glutathione and 3-mercaptopropionic acid to react with chromophore in its protein-bound state. These results confirm the hypothesis and stress the important role played by the negatively charged residues, especially the aspartic acid residues in the positions 33, 79 and 99 in guarding the chromophore from inactivation.
URI: http://hdl.handle.net/11455/15930
Appears in Collections:化學系所

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