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研究生:楊佰寯
研究生(外文):Pai-Chun Yang
論文名稱:探討人類細胞質蘋果酸酶和粒線體蘋果酸酶在異位調節位上的差異
論文名稱(外文):Determination of the differences in the allosteric site of the human c-NADP-ME and m-NAD(P)-ME
指導教授:洪慧芝洪慧芝引用關係
指導教授(外文):Hui-Chih Hung
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學系所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:70
中文關鍵詞:蘋果酸酶蘋果酸酶
外文關鍵詞:malic enzymeanalogues
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蘋果酸酶具有氧化去羧的功能,能催化蘋果酸轉換成丙酮酸和二氧化碳,並將NAD(P) +還原成NAD(P)H。這個催化的反應需要二價金屬離子(錳離子或鎂離子)的參與。在哺乳動物中,根據輔因子的專一性可以將蘋果酸酶分成三種異構物,分別是c-NADP-ME、m-NAD(P)-ME和m-NADP-ME。m-NAD(P)-ME是一種異位調節酵素,與受質蘋果酸結合後會具有正協同性,此外反丁烯二酸結合上異位調節位會造成異位活化的作用。為了探討c-NADP-ME和m-NAD(P)-ME在反丁烯二酸結合位上的差異,我們根據序列比對分析結果,在反丁烯二酸結合位上挑選第57、59、73和102號胺基酸,並且突變成相對應的胺基酸。在本篇研究中,我們將測試反丁烯二酸的結構異構物對這兩種蘋果酸酶的影響,並且研究這些胺基酸對c-NADP-ME三級結構的影響。從動力學結果得知,在野生型和突變型的c-NADP-ME並無明顯差異,代表替換掉Ser57、Asn59、Glu73和Ser102,並無法幫助我們成功的建立反丁烯二酸結合位。在m-NAD(P)-ME中,反式羧基的構型在酵素的異位調節活化作用中是重要的。若是雙羧基上接上乙基可能會形成異位調節抑制劑,進入反丁烯二酸結合位後,會抑制蛋白的活性。此外,從螢光的實驗中得知,突變c-NADP-ME反丁烯二酸結合位上的胺基酸會影響蛋白質三級結構的變化。總結以上的實驗,若突變反丁烯二酸結合位上的胺基酸對蛋白的活性、配合體(ligand)的結合和結構的變化都會一定程度的影響。

Malic enzyme catalyzes a reversible oxidative decarboxylation of L-malate into pyruvate and CO2, with concomitant reduction of NAD(P)+ to NAD(P)H. The reaction requires a divalent metal ion (Mn2+ or Mg2+) for catalysis. In mammals, malic enzymes have been divided into three isoforms according to their cofactor specificity as follows: c-NADP-ME, m-NAD(P)-ME, and m-NADP-ME. The m-NAD(P)-ME displays a positive co-operative manner of binding the substrate L-malate, and it can be allosterically activated by fumarate binding. To determine of the difference in allosteric site between human c-NADP-ME and m-NAD(P)-ME, The amino acid residues at positions 57, 59, 73 and 102 have been selected based on the sequence alignments, and substituted with corresponding residues. In this study, we investigated the effect of the structural analogues of the allosteric activator fumarate on human c-NADP-ME and m-NAD(P)-ME, and also we studied these residues effect in the tertiary structure of the c-NADP-ME. Our kinetic data clearly indicates that there was no significant difference in wild type and mutants of human c-NADP-ME. These results suggests that the substitution of Ser57, Asn59, Glu73, and Ser102 isn’t helpful in creation of fumarate binding site. In m-NAD(P)-ME, the dicarboxylic acid in a trans conformation around the carbon-carbon double bond is required for the allosteric activation of the enzyme. The dicarboxylic acid with ethyl group may be the allosteric inhibitor, which can enter the allosteric site to inhibit the enzyme activity. Furthermore, fluorescence studies of c-NADP-ME demonstrate that the mutants of fumarate binding site lead to the comformational changes of the tertiary structure. Our findings showed that the mutations at allosteric site has significant effects on the enzyme activity, ligand binding and structural coordination.

目錄………………………………………………………………………………........ I
縮寫表………………………………………………………………………………. III
表目錄………………………………………………………………………………. IV
圖目錄……………………………………………………………………………….. V
中文摘要……………………………………………………………………………. VI
英文摘要…………………………………………………………………………... VII
前言………………………………………………………………………………....... 1
實驗材料與方法……………………………………………………………………... 6
通透細胞的製備…………………………………………………………………… 6
定點突變法……………………………………………………………………….. 6
轉形作用………………………………………………………………………..… 8
質體的萃取……………………………………………………………………….. 8
重組蛋白質表現…………………………………………………………………… 8
蛋白質的純化……………………………………………………………………... 9
蛋白質的濃度測定……………………………………………………………….. 11
SDS-PAGE分析………………………………………………………………..… 12
酵素動力學分析………………………………………………………………….. 12
螢光光譜儀分析蛋白質結構的穩定性……………………………………………… 14
分析級超高速離心機分析四級結構……………………………………………...… 14
結果 ………………………………………………………………………………... 16
人類細胞質蘋果酸酶的酵素動力學參數…………………………………………… 16
反丁烯二酸和結構異構物對人類細胞質蘋果酸酶活化作用的影響…………………… 16
人類粒線體蘋果酸酶的酵素動力學參數…………………………………………… 17
反丁烯二酸和結構異構物對人類粒線體蘋果酸酶活化作用的影響…………………… 17
人類細胞質蘋果酸酶的三級結構穩定性…………………………………………… 19
人類細胞質蘋果酸酶和粒線體蘋果酸酶的四級結構變化………………………….… 20
討論…………………………………………………………………………………. 21
探討人類蘋果酸酶的異位調節位………………………………………………..… 21
反丁烯二酸和結構異構對人類細胞質蘋果酸酶的影響……………………………… 21
異位調節位在人類粒線體蘋果酸酶的活化特性…………………………………..… 23
雙羧基接上乙基造成抑制活性的作用……………………………………………… 24
突變人類細胞質蘋果酸酶的異位調節位造成三級結構的變化……………………..… 25
參考文獻………………………………………………………………………….… 28
附錄……………………………………………………………………………….… 61
主要儀器及器材………………………………………………………………..… 61
藥品試劑………………………………………………………………………… 62
相關藥品配製………………………………………………………………….… 64


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