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研究生:陳鴻萱
論文名稱:大腸桿菌Thioredoxinreductase的活性與連接兩個功能次單元的beta-sheet結構之關係
論文名稱(外文):The relation of thioredoxin reductase's activity and its B-sheet structure in Escherichia coli
指導教授:林苕吟
學位類別:碩士
校院名稱:國立交通大學
系所名稱:生物科技研究所
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:2002
畢業學年度:90
語文別:中文
中文關鍵詞:大腸桿菌黃質酵素
相關次數:
  • 被引用被引用:2
  • 點閱點閱:232
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大腸桿菌的Thioredoxin reductase是屬於黃質酵素中的pyridine nucleotide-disulfide oxidoreductase家族中的一員。此酵素為一個同型二聚體的蛋白質,每個單體分子量為35300道爾吞,分別包含了一個活性中心和輔基FAD,其催化的反應是利用NADPH將thioredoxin這個小蛋白質還原,電子傳遞的過程是由NADPH到FAD,再由還原態的FAD到thioredoxin reductase的活性中心,最後由此活性中心形成的dithiol和thioredoxin的雙硫鍵作用將它還原。從Thioredoxin reductase的結晶結構可看到此蛋白由兩個球狀次單元組成,且經由一個β-sheet結構所連接,其中,一個次單元含有一個輔基FAD,另一個次單元含有NADPH的結合位置與活性中心。當電子傳遞進行時,會有一個構形的轉變的發生:兩個次單元互相扭轉了66∘。而構形的改變需要一個旋轉的轉軸,即為連接兩個次單元的β-sheet結構,從文獻上已知Thioredoxin reductase構形的改變對其催化路徑是重要的,而我們推測β-sheet結構對其活性亦扮演關鍵性的角色。於是我們利用定點突變的方式改變此結構及靠近此結構的胺基酸,再測整體酵素的活性(Km及kcat值)。我們發現此結構上的突變對於整體活性的影響是顯而易見的,所以我們相信此β-sheet結構的確在大腸桿菌Thioredoxin reductase的催化路徑中扮演重要的角色。
中文摘要 …………………………………………………………………… i
英文摘要 …………………………………………………………………… ii
誌 謝 …………………………………………………………………… iii
目 錄 …………………………………………………………………… iv
表 目 錄 …………………………………………………………………… v
圖 目 錄 …………………………………………………………………… vi
附表目錄 …………………………………………………………………… viii
附圖目錄 …………………………………………………………………… ix
縮 寫 表 …………………………………………………………………… x
壹、 緒論 …………………………………………………………………… 1
1.1 Thioredoxin System …………………………………………… 1
1.2 Thioredoxin …………………………………………… 1
1.3 大腸桿菌的Thioredoxin ………………………………………… 2
1.4 大腸桿菌的Thioredoxin Reductase ……………………………… 2
1.5 研究目的 ……………………………………………………… 6
貳、 材料與方法 …………………………………………………………… 7
2.1材料 ……………………………………………………… 7
2.2方法 ……………………………………………………… 16
參、 實驗結果 ……………………………………………………………… 27
3.1利用PCR產生Thioredoxin reductase突變基因………………… 27
3.2 Thioredoxin reductase的質體架構 ……………………………… 27
3.3 Thioredoxin reductase突變基因的DNA定序 ………………… 27
3.4 Thioredoxin reductase的表現條件……………………………… 28
3.5 Thioredoxin reductase突變株的表現…………………………… 28
3.6 Thioredoxin reductase的純化…………………………………… 28
3.7 Thioredoxin的純化……………………………………………… 29
3.8突變株Thioredoxin reductase吸收光譜的測量……………… 30
3.9突變株Thioredoxin reductasedru最大吸收波長與吸光值的測定 30
3.10突變株Thioredoxin reductase的活性分析 …………………… 30
肆、 討論 …………………………………………………………………… 33
伍、 結論 …………………………………………………………………… 38
陸、 未來展望 ……………………………………………………………… 39
柒、 參考文獻 ……………………………………………………………… 40
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