四羧基苯基丙酮酸雙氧合酶 (HPPD ; 4-hydroxyphenyl- pyruvate dioxygenase) 催化酪氨酸代謝途徑中第二步驟，將 4-
hydroxyphenylpyruvate (HPP) 經氧化脫羧、取代基轉移及苯環氧化反應產生 homogentisate (HG)。 HPPD 屬於 α-keto acid 依賴型氧化酶家族中的一員。在人體中 HPPD 是一個雙聚體的蛋白，單體分子量為 43 kDa，共有 393 個氨基酸，反應時毎一單體都需要一個二價鐵作為輔因子。在此氧化酶家族酵素活性中心，均是以 2-His 1-carboxylate facial triad 的方式與金屬離子結合。本研究先由已知其他物種氨基酸序列及晶體結構比較，以及利用 DS modeling 模擬人類 4-HPPD 活性中心結構，從其中推測出與受質結合有關的 5 個氨基酸，Q334、F336、N363、F364 及 L367，再以定點突變的方式研究 HPPD 催化活性中心胺基酸突變對 HPP 受質催化反應的影響，來決定其結合位向。由 HPLC 分析產物 HG 發現，野生型 4-HPPD
比活性為 1.04 ± 0.12 nmole‧min/μg ， Km 為 0.19 mM ; N363V 突變型比活性剩 40% 為 0.41 ± 0.01 nmole/min/μg，Km 為 0.69 mM ，而 Q334L、F336L、L367K 突變型則測不到產物生成因此活性很低。以上 HPLC 分析結果也與菌株在培養基上表現型一致，僅有野生型及 N363V 在培養基上有酵素代謝後的紅棕色氧化產物。耗氧測定儀也得到相同的結果，野生型 4-HPPD對氧氣消耗比活性為 0.81 ± 0.18 nmole‧min/μg ； N363 突變型比活性剩 70% 為 0.55 ± 0.05 nmole/min/μg，而 Q334L、F336L、L367K 突變型則測不到氧氣的消耗。受質結合實驗，則可得到野生型 4-HPPD 與 4-HPP 解離常數 Kd = 0.14 ± 0.33 mM，而突變型 Kd 變大，酵素與受質結合能力變低。以上結果可看出，本研究在酵素活性中心所做的突變，皆對活性造成了很大的影響，且是在一開始受質結合就出了問題，可幫助我們了解其活性中心的結構，受質附近氨基酸要如何引導，而始有正確結合位向。

4-HPPD catalyzes the conversion of HPP to HG. This reaction is taken place in the second step of tyrosine catabolism, a reaction involving decarboxylation, substituent migration and aromatic oxygenation. 4-HPPD is member of α-ketoglutarate dependent oxygenase family requiring Fe (II) as a cofactor binding to active site in a way of 2-his- 1-carboxylate facial triad. Physiologically, it associates to form homo-
dimer. Each monomer has a molecular weight of 43 kDa with 393 amino acids. This research started with sequence alignments and crystal structures solved of HPPD between different species. Along with the human HPPD-HPP complex modeled by DS MODELING, We speculated five amino acids (Q334, F336, N363, F364 and L367) in C terminal substrate binding pocket related to substrate binding. Site directed mutagenesis at these positions was made to study the influences on enzyme activity and the substrate binding. From HPPD product, HG formation analyzed by HPLC, the specific activity and Km of HPPDWT are 1.04 ± 0.12 nmole‧min/μg and 0.19 mM ; HPPDN363V are 0.41 ± 0.01 nmole‧min/μg (60% decrease compared to WT) and 0.69 mM. The enzyme activity of other mutant protein was undetectable by HPLC. Which are consistent with the culture phenotype. Only HPPDWT and HPPDN363V colonies appeared red-brown color caused by the oxidized HG product. Others can not be seen with this phenomenon. The similar result is obtained by the oxygen consumption assay. The specific activity of HPPDWT on oxygen consumption is 0.81 ± 0.18 nmole‧min/μg and HPPDN363V is 0.55 ± 0.05 nmole‧min/μg (30% decrease compared to WT). Whereas no oxygen consumption observed on HPPDQ334L, HPPDF336L and HPPDL367K. The binding of HPP to HPPD was also examined by fluorescence quenching experiments. The dissociation constant, Kd of HPP to HPPDWT is 0.14 ± 0.33 mM, which is lower than other mutant proteins. It means the substrate binding capability of mutant proteins is weaker than wild type protein. Above all, the mutant protein constructed in this research do deteriorate the enzyme activity, especially in the substrate binding process. This research could help us to understand the structure of active site. And how could the residues in active site conduct the substrate to have the proper binding orientation.

序論 .................................................... 1
實驗材料 ................................................ 8
壹、藥品試劑 ........................................ 8
貳、主要儀器及器材 ................................. 10
參、試劑配置 ....................................... 11
實驗方法 ............................................... 15
壹、構築野生型人類重組 4-HPPD DNA .................. 15
一、設計引子 ................................... 15
二、限制酶(restrction enzyme)切目標DNA和載體 .. 15
三、DNA 膠體電泳 ............................... 16
四、回收 DNA ................................... 16
五、DNA 連結反應 (Ligation) .................... 17
六、轉殖 (transformation) ...................... 17
七、勝任細胞 (Competent Cell, DH5) 的製備 ..... 18
八、DNA 聚合酶連鎖反應 ......................... 19
貳、構築突變型人類重組 4-HPPD DNA .................. 20
一、設計引子 ................................... 21
二、DNA 聚合酶連鎖反應 ......................... 22
參、表現 4-HPPD 蛋白質 .............................. 24
肆、純化 4-HPPD 蛋白質 .............................. 25
伍、蛋白質含量測定 .................................. 27
陸、以 SDS-PAGE 鑑定蛋白質純度和分子量 .............. 27
柒、圓偏振二色旋分析光譜 (Circular Dichroism) ....... 28
捌、高效液態層析儀 (HPLC) ........................... 29
玖、耗氧測定儀 (Oxygraph) ........................... 31
拾、螢光減弱法分析 4-HPP 與 4-HPPD 之結合常數 ....... 32
拾壹、西方墨點法 (Western blotting) ................. 33
拾貳、DS modeling 模擬人類 4-HPPD 結構 .............. 35
結果 ................................................... 36
壹、野生型及突變型人類重組 4-HPPD DNA 構築 .......... 36
貳、野生型和變異型人類重組 4-HPPD 蛋白質純化 ........ 37
一、陰離子交換層析法 (Q Sepharose column) ....... 37
二、疏水性層析管柱 (Hydrophobic column) .......... 37
三、分子篩層析法 (S-100 column) ................. 38
四、人類野生型 4-HPPD 蛋白質 N 端定序 ........... 38
五、野生型與突變型人類 4-HPPD純化結果與西方墨點法
壓片 ........................................ 39
参、圓偏振二色旋光譜分析 ............................ 39
肆、野生型及突變型 4-HPPD 活性分析 .................. 39
一、利用高效液態層析儀 (HPLC) 分析反應產物 HG ... 39
二、耗氧測定儀 .................................. 41
伍、受質 4-HPP 滴定 4-HPPD .......................... 41
陸、人類 4-HPPD 結構模擬 ............................ 42
討論 ....................................................43
參考文獻 ................................................66

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