臺灣博碩士論文加值系統

人類肝臟四羥基苯基丙酮酸雙氧合酶 (4-HPPD；4-hydroxyphenylpyruvate dioxygenase)為參與酪氨酸分解途徑中的第二步驟，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）。人類4-HPPD是一個雙聚體的蛋白，每個單體在反應時都需要一個二價鐵作為輔因子，屬於α-KG家族其中的mononuclar non-heme iron(II)一類，這個家族酵素的鐵離子活性中心，多是以2-His-1-carboxylate facial triad方式與金屬離子結合。本研究先由已知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，再以定點突變分析突變蛋白對HPP基質催化反應及與鐵離子的結合常數。由HPLC方法分析，野生型4-HPPD對HPP親和性（Km）及催化常數（kcat）為5.18 μmol及0.0028 s-1，而突變型蛋白無法偵測產物HG的產生。耗氧測定儀所測得4-HPPD對4-HPP結合親和性（Km）值，H183A、H166A及E349A突變型蛋白比野生型分別增加約70、60及90倍。而在鐵離子滴定實驗中，突變型蛋白對鐵離子的結合常數（Kd）比野生型增加四至五倍。這些結果說明這三個與鐵離子結合胺基酸之重要性。這三個胺基酸側鏈的改變不僅影響與鐵離子的結合能力，也影響到酵素催化的活性，尤其在產物的步驟。

Human liver 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) catalyzes the second step reaction in tyrosine catabolism. It incorporates both atoms of oxygen into 4-hydroxyphenylpyruvate (4-HPP) to form homogentisate (HG), a reaction that involves decarboxylation, substituent migration, and aromatic oxygenation. 4-HPPD is an α-ketoglutarate / non-heme iron (II)-dependent enzyme that 4-HPP serves as both substrate and cosubstrate and ferrous iron binding the active site in a way of 2-His-1-carboxylate facial triad. Based on the amino acid sequence and structure alignments of 4-HPPD from different species, a structure of human 4-HPPD was modeled by DS modeling software. Three amino acids, H183, H266, and E349 coordinated with the iron ion in active site were identified. By using site-directed mutagenesis method, mutant recombinant 4-HPPD was cloned and expressed in E. coli. 4-HPPD is a homodimer composed mainly of β-sheet. Kinetic analysis of HPPD by HPLC method showed that the binding affinity of the wildtype is 5.1μΜ for HPP, but no any HG product was detectable from H183A, H266A, and E349A mutant proteins. Activity determinant by Oxygraph showed about 70, 60 and, 90 folds increasing in Km value to HPP for H183A, H266A, and E349A mutant protein, respectively. In addition, the titration experiment showed that the Kd values in ferrous ion binding for theses three mutants are about four to five folds higher than wildtype. From theses results, the three residues in the active site of HPPD is identified to be crucial for both iron binding and catalysis.

目 錄
序論 1
實驗材料 7
壹、藥品試劑 7
貳、主要儀器及器材 9
叁、試劑的配製 10
實驗方法 13
壹、選殖野生型及突變型人類重組4-HPPD DNA 13
貳、設計引子(Primer) 14
叁、DNA膠體電泳..............................................14
肆、限制酶(restriction enzyme)切目標DNA和載體 15
伍、回收DNA..................................................16
陸、DNA連結反應( Ligation) 16
柒、轉殖作用(Transformation) 17
捌、勝任細胞(Competent cell；DH5α)的製備 17
玖、表現4-HPPD蛋白質 18
拾、純化4-HPPD蛋白質 19
拾壹、蛋白質含量測定 21
拾貳、以SDS-PAGE鑑定蛋白質純度和分子量 22
拾叁、圓偏振二色旋 (Circular Dichroism)分析光譜 22
拾肆、高效液態層析儀 (HPLC) 23
拾伍、耗氧測定儀 .24
拾陸、鐵離子滴定 .25
拾柒、模擬人類4-HPPD結構 26
結果 27
壹、選殖野生型及變異型人類重組4-HPPD DNA 27
貳、野生型和變異型人類4-HPPD蛋白之純化 27
一、Q Sepharose column陰離子交換層析法 27
二、硫酸銨沉澱蛋白質 28
三、S-100 column分子過篩層析法 28
四、純化圖…………………………………………………………………28
叁、圓偏振二色旋光譜分析…………………………………………………28
肆、高效液態層析儀 (HPLC)………………………………………………29
伍、耗氧測定儀 (Oxygraph) 30
陸、鐵離子滴定 31
柒、人類4-HPPD結構模擬 31
討論 32
參考文獻 51

圖 目 錄

圖一、不同物種之4-HPPD胺基酸序列比較………………………………36
圖二、選殖變異型4-HPPD DNA 37
圖三、野生型4-HPPD經Q Sepharose管柱純化之SDS-PAGE圖…………38
圖四、野生型4-HPPD經S-100管柱純化之SDS-PAGE圖 38
圖五、H183A突變型4-HPPD經Q Sepharose管柱純化之SDS-PAGE圖…39
圖六、H183A突變型4-HPPD經S-100管柱純化之SDS-PAGE圖…………39
圖七、野生型及單點突變型4-HPPD純化圖………………………………40
圖八、野生型及雙點突變型4-HPPD純化圖………………………………40
圖九、野生型及突變型4-HPPD圓偏振二色旋光譜分析…………………41
圖十、HG標準曲線…………………………………………………………41
圖十一、高效液態層析儀偵測不同4-HPP基質濃度之反應情形…………42
圖十二、耗氧測定儀偵測不同4-HPP基質濃度之耗氧情形………………43
圖十三、耗氧測定儀分析不同4-HPP基質濃度下反應速率變化情形……43
圖十四、野生型及突變型4-HPPD在不同濃度鐵離子之色胺酸螢光光譜44
圖十五、野生型及單點突變型4-HPPD在不同濃度鐵離子下之百分比螢光變化44
圖十六、模擬人類4-HPPD結構……………………………………………45
圖十七、模擬之人類4-HPPD單體結構……………………………………45
圖十八、人類4-HPPD與抑制劑NTBC模擬之活性中心結構……………46
表 目 錄
表一、野生型及突變型4-HPPD圓偏振二色旋光譜分析 .47
表二、HPLC分析野生型及變異型4-HPPD酵素動力學 .48
表三、耗氧測定儀分析野生型及變異型4-HPPD酵素動力學 .49
表四、野生型及變異型4-HPPD與鐵離子結合常數(Kd)……………50


附錄

附錄一、4-HPPD催化反應 55
附錄二、酪胺酸代謝及發生酪胺酸血症途徑 55
附錄三、天然及合成之4-HPPD抑制劑 56
附錄四、4-HPPD催化機制 56
附錄五、阿拉伯芥4-HPPD的氫鍵網路 57
附錄六、mononuclear non-heme iron類酵素之催化途徑 57

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