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研究生:劉秀貞
研究生(外文):Liu,Hsiu-Chen
論文名稱:以定點突變方式探討人類重組四羥基苯基丙酮酸雙氧合酶活性中心與鐵離子結合胺基酸
論文名稱(外文):Studies of the iron-binding residues of human 4-hydroxyphenylpyruvate dioxygenase by site-directed mutagenesis
指導教授:李惠珍
指導教授(外文):Lee,Hwei-Jen
口試委員:周慰遠周記源
口試委員(外文):Chou,Wei-yuanChou,Chi-Yuan
口試日期:2012-07-25
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:91
中文關鍵詞:四羥基苯基丙酮酸雙氧合酶
外文關鍵詞:4-hydroxyphenylpyruvate dioxygenase
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四羧基苯基丙酮酸雙氧合酶 (4-HPPD ; 4-hydroxyphenyl- pyruvate dioxygenase;EC 1.13.11.27)它催化酪氨酸代謝途徑中第二步驟。4-HPPD催化4-hydroxylphenylpyruvate(4-HPP)經氧化脫羧、取代基轉移及苯環氧化步驟產生Homogentisate(HG)。4-HPPD 屬於α-keto acid 依賴型非血基質需鐵氧化酶家族,此家族酵素的鐵離子活性中心,大多是以2-His-1-carboxylate facial triad方式與金屬離子結合。首先,本研究與其他物種胺基酸序列及晶體結構比較,以及利用Discovery Studio軟體模擬人類4-HPPD結構,確認活性中心與鐵離子結合的三個胺基酸為H183、H266及E349,再以定點突變的方式研究此三個胺基酸在催化作用上扮演重要的角色。利用圓偏振二色旋分析,發現野生型和突變型有類似的二級結構。利用耗氧測定儀和HPLC研究野生型4-HPPD比活性,分別為3±0.51nmole/min/μg及3.15±0.2nmole/min/μg。耗氧測定儀分析,突變型H183A、H266A、E349D、E349G、E349Q的比活性為野生型之2.1%、10.7%、19.3%、2.7%、17.7%;HPLC分析,突變型H183A 、E349D、E349G的比活性為野生型之0.64%、16.4%、2.6%,明顯下降。在HPP酵素動力學分析,發現E349Q之Km增加5倍,所有突變蛋白在kcat/Km是明顯降低。Fe2+ 之酵素動力學,則H266A及E349Q之Km值與野生型相比較分別增加21倍及12倍,所有突變蛋白之kcat值和kcat/Km是明顯降低。突變H266A之產物分析除HG外,另有HPA產物產生,說明,uncouple反應發生。此研究證實這三個胺基酸對鐵離子結合及催化作用上分別扮演不同的重要的角色。
4-hydroxyphenylpyruvate dioxygenase(4-HPPD; EC 1.13.11.27) catalyzes the second step in the pathway for the catabolism of tyrosine.
It incorporates both atoms of oxygen into 4-hydroxyphenylpyruvate to form homogentisate, a reaction that involves decarboxylation, substituent migration , and aromatic oxygenation. 4-HPPD belongs to the alpha-keto acid-dependent subgroup of non-heme dioxygenases. A ferrous iron binds in the active site in a way of 2-His-1-carboxylate facial triad. Alignments of amino acid sequence and crystal structure confirmed that H183, H266 and E349 coordinated with ferrous ion in active site. To understand the role of three amino acid site-directed mutagenesis was investigated. Wild-type and mutant 4-HPPD have similar secondary structure as analysed by CD. The specific activities of wild-type HPPD was determined to be 3±0.5 nmole/min/μg and 3.2±0.2 nmole/min/μg of by oxygraph and HPLC assay, respectively. The specific activity of mutant of H183A、H266A、E349D、E349G、E349Q have about 2.1%、10.7%、19.3%、2.7%、17.7% , Mutant of H183A、E349D、E349G compared with wild-type have about 0.64%、16.4%、2.6% by oxygraph and hplc assay, respectively. The HPP kinetic analysis showed increased Km value for E349Q.The kcat and kcat /Km were significantly decreased as compared to these of wild-type enzyme. The Km value of Fe2+ for H266A and E349Q were 21-fold and 12- fold increased .The kcat and kcat /Km were significant decreased as compared to wild-type enzyme. In addition , HPA product were detected for H266A indicate the uncouple rex. This study indicates the crucial and distinct role of the three amino acids in ferrous binding and catalysis.


總目錄

目錄 I
圖目錄 III
表目錄 V
附錄 VI
名詞縮寫對照表 VII
中文摘要 VIII
英文摘要 X

目錄
緒論 1
實驗材料 7
壹、 藥品試劑 7
貳、 主要儀器及器材 9
參、 試劑配製 10
實驗方法 15
壹、 構築突變型人類重組 4-HPPD DNA : 15
一、 設計(primer)引子 15
二、DNA 聚合酶連鎖反應(PCR) 16
貳、 細菌培養 17
一、勝任細胞 (Competent Cell, DH5α) 的製備: 17
二、轉殖 (transformation): 18
參、蛋白質(4-HPPD)表現 19
肆、蛋白質(4-HPPD)純化 20
一、 破菌: 20
二、陰離子交換層析管柱純化 20
三、疏水性層析管柱(Sourse-PHe column; 1.6cmX 90cm) 20
四、分子篩層析管柱(Gel filtration S-100 ; 2.5 cm X 90 cm) 21
五、濃縮 21
伍、蛋白質分析 21
陸、SDS-PAGE 鑑定蛋白質純度及分子量 22
一、膠體配置 22
柒、高效液態層析儀 (HPLC) 23
一、利用高效液態層析儀及反向層析 C18 管柱 23
二、 野生型及突變型 4-HPPD 在230nm分析: 24
捌、耗氧測定儀 (OXYGRAPH) 25
玖、西方墨點法 (WESTERN BLOTTING) 26
一、膠體配置: 26
二、轉漬(Transfer): 27
三、阻塞(Blocking): 27
四、次日加入一級抗體(Primary antibody detection): 27
五、清洗(Wash): 27
六、加入二級抗體(Secondary antibody detection): 28
七、清洗(Wash): 28
八、顯示(ECL detection): 28
拾、圓偏振二色旋分析光譜 (CIRCULAR DICHROISM) 28
一、圓偏振二色旋分析光譜主要用於分析各種蛋白質之二級結構: 28
二、圓偏振二色旋分析光譜 (Circular Dichroism)參數設定: 29
三、用DichroWeb的網站分析蛋白質α-Helix、β-Sheet、random coil 所占比例: 29

實驗結果 31
壹、 野生型和突變型人類重組 4-HPPD 蛋白質純化 31
一、 陰離子交換樹脂層析法(Q Sepharose chromatography) 31
二、疏水性層析管柱(Hydrophobic Interaction Chromatography) 31
三、分子篩層析管柱(S-100 column) 32
貳、 野生型與突變型人類4-HPPD 純化結果以 SDS-PAGE與西方墨點法 33
參、 圓偏振二色旋光譜分析 33
肆、野生型及突變型 4-HPPD 活性分析 33
一、耗氧測定儀 ( Oxygraph )分析催化過程中之氧氣消耗量: 33
二、 利用高效能液態層析儀 (HPLC) 分析建立標準曲線 36
三、 利用高效能液態層析儀 (HPLC)在230nm下分析野生型和突變型4-HPPD催化反應產物 HG和HPA生成: 38
討論 39
參考文獻 77

圖目錄
圖一、野生型4-HPPD純化後SDS-PAGE電泳分析圖 43
圖二、野生型人類重組4-HPPD純化後SDS-PAGE電泳分析圖 44
圖三、突變型人類重組4-HPPD通過不同Column純化後,可發現突變型人類重組4-HPPD純度越來越高。 45
圖四、野生型與突變型人類4-HPPD利用西方墨點法及冷光儀測定 47
圖五、(A)野生型與突變型4-HPPD蛋白質經純化後以SDS-PAGE進行分析其純度。 48
(B) 野生型與突變型4-HPPD蛋白質經純化後以西方墨點法進行分析。 48
圖六、利用圓偏振二色旋分析光譜分析野生型和突變型4-HPPD之二級結構。 49
圖七、利用SELCON3的方法,FIT出野生型和突變型4-HPPD理想圖和實際值的狀況。 50
圖八、利用Oxygraph分析野生型與突變型人類重組4-HPPD之耗氧量。 51
圖九、野生型和突變型4-HPPD以Oxygraph分析基質HPP之酵素動力學 52
圖十、野生型和突變型4-HPPD以Oxygraph分析在不同濃度FE2+之酵素動力學 53
圖十一 : 以HPLC分析在波長288nm不同HG濃度,沖堤位置及所呈現不同吸收波。 54
圖十二: HG在波長288nm定量標準曲線 55
圖十三、HPLC分析野生型和突變型人類重組4-HPPD之比活性比較圖 56
圖、十四:以HPLC分析在波長230nm下,所呈現之HG面積 57
圖十五 :HG在波長230nm定量標準曲線 58
圖十六:以HPLC分析在波長230nm下(不加過氯酸),所呈現之HPA面積。 59
圖十七 :HPA在波長230nm定量標準曲線 60
圖十八:在波長230nm下偵測,CONTROL組(反應劑中不加HPPD) 61
圖十九:在波長230nm下偵測,用1.5μg野生型4-HPPD分析,產生HG和HPA,三重覆。 61
圖二十:在波長230nm下偵測,用15μg突變型H183A分析,不加過氯酸,產生HG和HPA,二重覆。 62
圖二十一:在波長230nm下偵測,用20μg突變型H266A分析,不加過氯酸,產生HG和HPA,二重覆。 62
圖二十二:在波長230nm下偵測,用20μg突變型E349A分析,不加過氯酸,產生HG和HPA,二重覆。 63
圖二十三:在波長230nm下偵測,用5μg突變型E349D分析,不加過氯酸,產生HG和HPA,二重覆。 63
圖二十四:在波長230nm下偵測,用15μg突變型E349G分析,不加過氯酸,產生HG和HPA,二重覆.。 64
圖二十五:在波長230nm下偵測,用20μg突變型E349Q分析,不加過氯酸,產生HG和HPA,二重覆。 64
圖二十六、不同物種之4-HPPD胺基酸序列比對。 代表與鐵離子結合的三個胺基酸位置。 65


表目錄
表一、 野生型人類重組 4-HPPD 之純化表 66
表二、野生型及突變型4-HPPD之二級結構分析 67
表三、野生型及突變型4-HPPD以Oxygraph分析求酵素比活性 68
表四、 野生型及突變型4-HPPD 以Oxygraph分析基質HPP之動力學參數 69
表五、野生型及突變型4-HPPD對不同金屬之酵素比活性 70
表六、野生型及突變型4-HPPD以Oxygraph分析在不同濃度Fe2+之酵素動力學參數 71
表七、野生型與突變型 4-HPPD 以 HPLC 求酵素比活性 72
表八、在波長230NM下,偵測野生型及突變型4-HPPD分別產生HG和HPA百分比、活性比 73


附錄
附錄一、酪胺酸代謝途徑及其代謝基因缺陷所引發之疾病 74
附錄二、Order bi-bi steady state kinetics 75
附錄三、4-HPPD 催化途徑 76












參考文獻

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