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Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22458
標題: 人類粒線體蘋果酸酶異位調節區中第102號天門冬胺酸和第98號精胺酸間的交互作用對酶活性調節機制之探討
Characterization of the interactions between Asp102 and Arg98 in allosteric site involving the regulatory mechanism of human mitochondrial NAD(P)+-dependent malic enzyme.
作者: 何珮慈
Ho, Pei-Tzu
關鍵字: malic enzyme;蘋果酸酶偶合能;coupling energy;mutation cycle;突變循環
出版社: 生命科學系所
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摘要: 
人類粒線體蘋果酸酶 (EC 1.1.1.39) 在結構上是由四個完全相同的單體所組成之四聚體，可分為較緊密的雙聚體界面及較鬆散的四聚體界面，而每個單體都具有各自的活性中心，能將蘋果酸及NAD(P)＋在二價金屬離子(如錳、鎂離子)的催化下，進行氧化脫羧作用，形成產物丙酮酸、二氧化碳及NAD(P)H；人類粒線體蘋果酸酶的活性會分別受到反丁烯二酸及腺苷三磷酸的活化及抑制，目前研究顯示反丁烯二酸為異位活化劑而腺苷三磷酸則為競爭型抑制劑。由晶體結構證實，除了活性中心之外，人類粒線體蘋果酸酶在雙聚體界面處還具有異位調節區的存在，而反丁烯二酸所扮演的異位活化劑角色，其活化機制可能為促使四聚體之重排。在先前的研究中指出Asp102對於反丁烯二酸在異位調節區的活化是一個必要的胺基酸， Asp102突變後對於反丁烯二酸的結合有很劇烈的影響。Arg98的側鏈就在Asp102旁邊，因此推測Arg98 與Asp102之間可能存有吸引力，使得Arg98之胺基酸側鏈改變時，透過與Asp102之作用力，間接影響到反丁烯二酸的作用。在比對不同種生物體內之蘋果酸酶胺基酸序列後發現，Arg98具有高度保留性，因此針對98和102這兩個位置之胺基酸作定位突變。研究中利用雙突變循環 (double mutant cycle) 的方法來分析這兩個胺基酸之間的交互吸引力，進ㄧ步了解兩者之間的關係。
從實驗中發現，將Arg98的正電荷去掉之後，會使活化倍率大幅的下降，由KA的結果推測，可能是因為反丁烯二酸的結合更緊，結構上無法自由的轉動，使得反丁烯二酸無法藉由此胺基酸達到調控活化的功能。由雙突變循環分析證實Arg98和Asp102之間是相互依賴的，所得到之偶合能量有-3.52 kcal/mol之多。結合酶動力學、熱力學及晶體結構結果，證實了Arg98在人類粒線體蘋果酸酶異位調節區中，亦扮演極重要之角色，即使並非直接與反丁烯二酸產生鍵結，可能藉由與Asp102的作用，間接影響反丁烯二酸的親和力。

Human mitochondrial NAD(P)+-dependent malic enzyme (EC 1.1.1.39) is a homotetrameric protein. This homotetramer is a double dimer structure and the dimer interface is more intimately contacted than the tetramer interface. Every monomer has its own active site and catalyzes a reversible oxidative decarboxylation of L-malate to give carbon dioxide and pyruvate in the concomitant reduction of NAD(P)+ to NAD(P)H. Human mitochondrial NAD(P)+-dependent malic enzyme is an allosteric enzyme. Fumarate acts as an activator and ATP acts as a competitive inhibitor. Fumarate is bound at the dimer interface about 30 Å away from the active site, confirming that fumarate functions through an allosteric mechanism. Previous studies have indicated that Asp102 is an essential residue in the activation by fumarate at the allosteric site of the enzyme. Mutation of Asp102 caused a drastic effect on fumarate binding to the enzyme. The side chain of Arg98 is located next to Asp102. Since Asp102 and Arg98 are highly conserved in malic enzyme of most species, we used a double-mutant cycle to study investigate possible interactions between these two residues. Eleven mutants were created and the corresponding kinetic parameters were derived to analyse the free energy change and the coupling energy (ΔΔGint) between any two residues.
The activation fold, however, was significantly reduced by eliminating the positive side-chain of Arg98. A coupling energy of -3.52 kcal/mol was obtained for Asp102 and Arg98. Based on combined the kinetic data and structural information, we suggest that the balance of electric charge at allosteric site is essential for enzyme activation and the interaction between Asp102 and Arg98 has significant effects on fumarate activation. Our data indicate that Arg98 is important for fumarate binding even though it does not directly interact with fumarate. It seems to change the affinity of the enzyme with fumarate via its effect upon Asp102.
URI: http://hdl.handle.net/11455/22458
其他識別: U0005-2906200602340400
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