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研究生:蔡東圻
研究生(外文):Tung-Chi Tsai
論文名稱:人類重組四羥基苯基丙酮酸雙氧合酶酵素催化與基質結合之影響之探討
論文名稱(外文):The Study of the Effect of Amino Acid on the Binding and Catalytic Function of Human 4-hydrophenylpyruvate dioxygenase
指導教授:李惠珍李惠珍引用關係
指導教授(外文):Lee Hwei-Jen
口試委員:張永龍黃啟清
口試委員(外文):Chang, Yung-LungHwang Chi-Ching
口試日期:2019-05-15
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:2019
畢業學年度:107
語文別:中文
論文頁數:93
中文關鍵詞:人類重組四羥基苯基丙酮酸雙氧合酶
外文關鍵詞:4-hydrophenylpyruvate dioxygenase (HPPD)
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四羥基苯基丙酮酸雙氧合酶 (4-HPPD) 屬於非血基質需鐵之α-酮酸依賴型氧化酶家族,其活性中心以2-His 1-carboxylate facial triad的方式與二價鐵離子結合。催化四羥基苯基丙酮酸 (4-HPP) 經氧化脫羧、取代基轉移和苯環氧化反應產生黑尿酸 (HG),此催化反應也為酪胺酸代謝途徑中的第二步驟。本研究將探討酵素活性中心的胺基酸F359L、F364I、F368Y及F364I/F368Y,經突變後對基質與酵素的結合是否造成影響,並以酵素活性分析、基質結合與產物生成進行分析。酵素活性分析中,利用Oxygraph assay進行反應時含氧變化的分析酵素催化動力學,發現F359L、F364I及F364I/F368Y的Km值較野生型各下降約8倍、6倍及4倍,而F368Y與野生型差異不大;kcat/Km則是F359L和F368Y與野生型相近,F364I和F364I/F368Y則為野生型的約3倍和2倍,且皆有發現基質抑制的現象。利用HPLC分析反應後的產物HG生成,可發現F359L、F364I及F364I/F368Y的催化比活性較於野生型各下降約6.5倍、1.6倍及6.5倍,F368Y則較於野生型上升約2倍。等溫滴定量熱法的結果中顯示4-HPPD與Co2+複合物與4-HPP之結合為放熱反應,將Kd值與野生型比較後發現,F364I/F368Y上升約2.6倍而F359L、F364I與F368Y則下降約2倍、5倍與3倍。從結果來看,F359L、F364I、F368Y和F364I/F368Y的突變影響酵素與基質之結合親和性,尤其是受與基質芳香環支鏈之π鍵交互作用的影響,導致催化反應之基質轉換速率及Km值皆下降,進而產生基質抑制之現象。
4-Hydroxyphenylpyruvate dioxygenase (4-HPPD) is a nonheme iron/α-oxoacid-dependent dioxygenase that have the common 2-His 1-carboxylate facial triad for iron binding. 4-HPPD catalyses the second step in tyrosine catabolism involving conversion of 4-hydroxyphenylpyruvate (4-HPP) to homogentisate (HG) via steps of decarboxylation, substituent migration and aromatic hydroxylation. We investigated the effect of F359L, F364I, F368Y and F364I/F368Y mutants on the substrate binding and catalytic activity of 4-HPPD. The activity was analyzed using the Oxygraph assay and HPLC to determine the oxygen consumption and HG product formation in the reaction, respectively. It found that the specific activity of the mutant F359L, F364I and F364I/F368Y was reduced by 6.5-, 1.6- and 6.5-fold, respectively, F368Y was increased by 2-fold, as compared with that of the wild type. And the mutant F368Y is similar to the wildtype. Compared with wild type, the Km values for F359L, F364I and F364I/F368Y were reduced by 8-, 6- and 4-fold, respectively. And the mutant F368Y is similar to the wildtype. The kcat/Km values of F359L and F364I/F368Y are similar to that of the wild type, however, F364I and F364I/F368Y was increased by 3-, 2-fold. Substrate inhibition were observed in kinetic analysis for the three mutants. Isothermal titration calorimetry analysis showed that the binding of 4-HPP by 4-HPPD-Co2+ complex was an exothermic reaction. The Kd value of F364I/F368Y was increased by 2.6-fold, F359L, F364I and F368Y was reduced by 2-, 5- and 3-fold, as compared with that of the wild type. The results suggested that the mutants of F359L, F364I, F368Y and F364I/F368Y affect the binding of substrate, in particular the π-π interactions between the aromatic sidechain of substrate and these residues. It might cause the substrate inhibition behavior from reduced substrate turnover rate and Km values in the catalytic reaction of the mutant enzymes.
目錄 I
圖目錄 III
表目錄 V
附錄目錄 VI
名詞縮寫對照表 VII
中文摘要 VIII
英文摘要 IX
緒論 1
壹、 酪胺酸 (Tyrosine) 相關疾病 2
貳、 4-HPPD的結構 3
參、 催化反應 7
肆、 自身氫氧化反應 8
伍、 催化反應機制 9
研究目的 11
實驗材料 12
壹、 藥品 12
貳、 主要儀器及器材 14
參、 試劑配製 15
實驗方法 20
壹、 取得突變型人類重組 4-HPPD DNA 20
貳、 表現4-HPPD野生型與突變型蛋白質 23
參、 4-HPPD 活性分析方法 27
結果 36
壹、 野生型及突變型蛋白質純化 36
貳、 圓偏振二色旋光譜分析 38
參、 比活性分析 39
肆、 基質4-HPP之酵素動力學活性分析 40
伍、 基質結合位置分析 41
陸、 Ligand對Buffer的稀釋熱 42
柒、 ITC 實驗結果 42
討論 44
參考文獻 87

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