臺灣博碩士論文加值系統

人類粒線體蘋果酸酶（EC 1.1.1.39）催化氧化脫羧反應是以二價金屬離子作為輔因子，催化蘋果酸分解成為丙酮酸及二氧化碳，並且伴隨著將輔酶 NAD(P)+ 還原成為NAD(P)H。且其活性會受到反丁烯二酸和腺苷三磷酸的調控，屬於異位調節酶；反丁烯二酸為活化劑而腺苷三磷酸為抑制劑。在結構上，蘋果酸酶是由四個完全相同單元體所組成的四聚體。由解出酶與NAD+複合物的結晶結構得知每個單元體中含有兩分子的NAD+，分別位於活性中心及異位調節區；另外酶與腺苷三磷酸複合物的晶體結構則顯示了腺苷三磷酸腺的結合位置與NAD+的相同。因腺苷三磷酸亦可於活性中心佔據NAD+的結合位置而可能直接影響酶活性，而當腺苷三磷酸於異位調節區結合時，如何調控酶功能則有待釐清。本研究設計利用針對腺苷三磷酸相關位置（R197及R542）以定點突變的方式改變為其他胺基酸，配合動力學、圓偏振二旋光譜分析和分析級超高速離心等研究工具，探討腺苷三磷酸對人類粒線體蘋果酸酶所可能造成的活性影響以及結構變化。結果顯示，腺苷三磷酸確實會抑制酶活性，對蘋果酸及NAD+競爭均屬於競爭性抑制現象。由分析級超高速離心的分析結果得知，當腺苷三磷酸存在時會誘使酶結構趨向形成四聚體。因此，腺苷三磷酸可能作用於異位調節區，藉此調控人類粒線體蘋果酸酶的四級結構，而達到抑制酶活性的目的。

Human mitochondrial NAD(P)+-dependent malic enzyme（EC1.1.1.39, mNAD-ME）catalyzes the oxidative decarboxylation of malate to pyruvate and CO2 with the concomitant reduction of NAD(P)+ to NAD(P)H. The enzyme also requires divalent cation, Mn2+ or Mg2+, as an essential cofactor. The catalytic activity is allosterically regulated by fumarate as an activator and by ATP as an inhibitor. The enzyme is a homotetrameric protein with four monomers arranged in the corners of the planar. The first solved crystal structure of mNAD-ME in complex with NAD+ and has two NAD+ molecule bound per monomer. These two sites are designated as the active site and allosteric site, respectively. Subsequently the structure of mNAD-ME-ATP complex has also be solved, that the binding site of ATP is in the active site as well as in the allosteric site. In the thesis, we tried to elucidate the effect of ATP in the allosteric site that binds the second molecule of NAD+. We used site-directed mutagenesis to mutate two residues in the ATP-binding site （R197 and R542）. We examined the functional properties of the mutants by kinetic analysis and structural changes was assessed by circular dichroism and analytical ultracentrifuge. The results indicated that ATP was a competitive inhibitor with respect to malate and NAD+. The results from analytical ultracentrifuge indicated that ATP promoted tetramer formation.

頁次
目錄…………………………………………………………………….………I
縮寫表………………………………………………………………………………II
表目錄………………………………………………………………….……III
圖目錄………………………………………………………………………………IV
中文摘要………………………………………………………………………………VI
英文摘要………………………………………………………………………….…VII
壹、前言………………………………………………………………………….……1
貳、實驗材料與方法
一、藥品試劑………………………………………………………………….6
二、培養液及緩衝液之製備…………………………………………...……8
三、主要儀器及器材……………………………………………………………………...…….11
四、實驗方法
（一）、突變型人類粒線體蘋果酸酶質體製備……………………………12
（二）、重組野生型及各突變型人類粒線體蘋果酸酶之純化……....…16
（三）、人類粒線體蘋果酸酶活性分析……………………………………20
（四）、人類粒線體蘋果酸酶之結構分析………………………..………21
參、結果………………………………………………………………………………23
肆、討論………………………………………………………………………………27
伍、參考文獻………………………………………………………………………………33
表………………………………………………………………………………37
圖………………………………………………………………………………44

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