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研究生:徐于婷
研究生(外文):Yu-Ting Hsu
論文名稱:耐輻射奇異球菌天門冬胺酸轉胺酶之生化研究
論文名稱(外文):Biochemical Studies on the Aspartate Aminotransferase from Deinococcus radiodurans R1
指導教授:張文章
指導教授(外文):Wen-Chang Chang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:80
中文關鍵詞:天門冬胺酸轉胺酶耐輻射奇異球菌
外文關鍵詞:AspATAminotransferaseradioresistanceDeinococcus radiodurans R1
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耐輻射奇異球菌(Deinococcus radiodurans R1;D. radiodurans R1)對於環境中的游離輻射、UV輻射與過氧化氫的抵抗力極高。瞭解D. radiodurans R1所擁有的特殊分子及生理層次可以幫助我們認識生物體應對輻射傷害而發展的耐受機制。本研究主要為探討耐輻射奇異球菌之天門冬胺酸轉胺酶 (Aspartate Aminotransferase;AspAT;EC 2.6.1.1)的各項性質。
天門冬胺酸轉胺酶也稱為麩胺酸-草乙酸轉胺酶(Glutamate Oxaloacetate Transaminase;GOT)。它催化麩胺酸與天門冬胺酸之間的轉胺反應,與氮的循環以及能量利用有密切關係。在耐輻射奇異球菌中,AspAT的基因稱為aspC,約1167 bp,轉殖於大腸桿菌進行表現之後,使用金屬親和性層析 ( Ni-NTA affinity chromatography ) 純化達到均質。再以Factor Xa切除Fusion Protein ( Maltose-binding Protein ),並由第二次的金屬親和性層析純化得到DrAspAT。此時蛋白質的比活性為104.6 U/mg。以逆相HPLC去除二次純化後均質所含鹽類,經質譜儀測定分子量,其值與理論值完全相符。
DrAspAT與PcAspAT (豬的細胞質)都有明顯耐熱性,至80 ℃依然還有50 % 的殘留活性。然而在X-ray輻射試驗中,DrAspAT顯示出較強的耐輻射性,且兩酵素的損害程度皆與成濃度反比。
另一方面,利用Program O模擬DrAspAT的結構以預測DrAspAT結構中重要殘基的分布,選取其中六個殘基進行定點突變,所得的突變株經酵素分析後結果都無活性殘留,證實此一模擬的結構可信度極高。
模擬所得DrAspAT結構相較於嗜中溫生物體之AspAT而言,大體相似。然而在N端的序列結構與辨認酸性基質殘基的改變,顯示出兩類生物在演化上的分歧。比對序列後發現,組成酵素的胺基酸成分似乎與其生存環境相關。DrAspAT序列中有高比例可加強構造穩定性的Proline殘基以及不含易使酵素分子被含氧自由基攻擊的Cystein 殘基存在。
透過DrAspAT的生化研究,再加上目前可獲得的研究資訊,可以一窺耐輻射奇異球菌生存策略與蛋白質分化機制的奧秘。
Deinococcus radiodurans R1 is well-known for its extraordinary resistance against high-dose ionizing radiation, UV radiation and hydrogen peroxide. It is an ideal target for the study of survival strategies adopted by extremophiles under various harsh environmental conditions. This thesis aims at the characterization of an enzyme, aspartate aminotransferase (AspAT, EC 2.6.1.1), from D. radiodurans R1.
Aspartate aminotransferase, also known as glutamate oxaloacetate transaminase (GOT), catalyzed the interconversion between glutamate and aspartate. It is involved in the metabolism of nitrogen-containing biomolecules and is essential for energy metabolism. In D. radiodurans R1 AspAT is encoded by the gene aspC of about 1167 bp. AspAT from D. radiodurans R1 has been cloned, expressed in E. coli, purified and characterized. The expressed recombinant protein was purified to apparent homogeneity by Ni-NTA affinity chromatography. The fusion protein (Maltose-binding Protein) was removed by Factor Xa. The cleavage product was further purified by Ni-NTA column again to yield a homogeneous protein. Its molecular mass was confirmed by mass spectral analysis after desalting by reversed-phase HPLC through a C8 column. The purified protein had a specific activity at 104.6 U/mg. This enzyme, designated as DrAspAT, exhibits remarkable thermal tolerance and radioresistance, as compared with AspAT from porcine cytoplasmic. The radioresistance is dependent on protein concentration: the higher the protein concentration, the less damage caused by X-ray.
The molecular structure of DrAspAT was modeled by using Program O and a template structure of Thermus thermophilus AspAT (1bjw; TtAspAT ). Several catalytically important residues have been indenfied from the modeled strucute. In order to test the modeled structure site-directed mutagenesis experiment was carried out on those important residues. The results showed that all the mutant enzymes lost the enzymatic activity, thus confirming the important roles they play in catalysis.
Sequence alignment and analysis showed that DrAspAT is similar to the AspATs from mesophilic organisms in conformation. However, the comparison result showed evolution diversity between those two kinds of AspATs by the obvious difference in the N-terminal sequence and the residues recognizing the distal carboxylate group of the substrate. Moreover, by advanced analysis for the composition of amino acids, DrAspAT contain higher percentage of proline residues and no cysteine in contrast to other mesophilic counterpars, this preference of DrAspAT may partially account for its remarkable structural stability.
As a result, analysis for biochemical studies on DrAspAT with other data about AspATs from various species could give us some clues about extraordinary radioresistance of D. radiodurans R1 and evolution direction of proteins.
表目錄.. 5
圖目錄.. 6
摘要.... 8
Abstract 9
第一章 緒 論 11
第一節 轉胺酶總論 11
一 轉胺酶 11
1. 轉胺作用 11
2. 轉胺酶分類 13
二 天門冬胺酸轉胺酶 15
1. 分類 15
2. 功能 16
3. 催化機制 16
4. 結構 19
第二節 耐輻射奇異球菌 21
一 前言 21
二 適應性輻射演化 21
1. 普遍適應 22
2. 環境改變 22
3. 群島效應 22
三 嗜極菌 22
四 耐輻射奇異球菌發現歷史 23
五 耐輻射奇異球菌生理構造與遺傳物質 23
六 耐輻射性 25
1. 基因體 25
2. 物理防護 26
3. 金屬離子 26
第三節 研究目的與研究流程 28
一 應用 28
1. 製備胺基酸及胜肽 28
2. 藥物 28
3. 廢料清除 28
二 研究目的 29
1. DrAspAT定性 29
2. AspAT比較 29
3. DrAspAT之功能性殘基確認 29
4. 耐輻射奇異球菌抗輻射性的相關因子 29
三 研究流程 30
第二章 天門冬胺酸轉胺酶表現純化與特性分析 31
第一節 前言 31
第二節 材料與方法 34
一 聚合酵素鏈鎖反應 34
二 限制酶切反應 35
三 接合反應 36
四 轉形反應 36
五 DrAspAT表現 37
六 超音波破菌 37
七 蛋白質膠體電泳分析 38
八 鎳金屬親和管柱層析分析 39
九 接合蛋白質切除反應 40
一 蛋白質定量分析-Bradford法 40
一一 活性分析-MDH-coupling assay 41
一二 高壓液相層析與質譜分析 42
一三 西方點墨法 42
一四 熱穩定性試驗 43
一五 儲存條件試驗 43
一六 基質專一性試驗 44
一七 X-ray 輻射試驗 44
第三節 結果與討論 45
一 結果.. 45
二 討論.. 60
1. aspC基因轉殖 60
2. DrAspAT之表現 60
3. DrAspAT之純化 60
4. DrAspAT確認 61
5. 儲存條件 61
6. 基質專一性分析 62
7. 熱穩定性 62
8. X-ray 輻射耐受性 63
第三章 天門冬胺酸轉胺酶比對與分子模擬 64
第一節 前言 64
第二節 原理與方法 66
一 天門冬胺酸轉胺酶之比對分析 66
二 同源分子模擬 67
1. 原理 67
2. 方法與步驟 67
三 DrAspAT定點突變 68
第三節 結果與討論 69
一 結果 69
二 討論 76
1. AspATs 比對分析 76
2. 分子模擬 77
3. 突變酵素分析 77
第四章 結 論 與 展 望 78
第一節 DrAspAT之生化研究 78
第二節 DrAspAT之結構分析 79
第三節 展望 80
附錄一:儀器設備一覽表
附錄二:縮寫表
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