Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23743
標題: 探討選擇性抑制劑對人類粒線體蘋果酸酶的影響
Study on the specific inhibitors of human m-NAD(P)+-ME
作者: 李紹瑜
Li, Shao-Yu
關鍵字: malic enzyme
蘋果酸酶蛋白結構
protein structure
specific inhibitor
專一性抑制劑
出版社: 生命科學系所
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摘要: 蘋果酸酶(malic enzyme,ME)以錳或鎂等二價金屬離子為輔因子,使蘋果酸氧化脫羧形成丙酮酸與二氧化碳,同時伴隨NAD(P)H生成。其廣泛存在於生物體中且胺基酸序列保留度高。在哺乳類動物中依運用的輔酶與在細胞中位置不同,可分為三種異構型c-NADP+ -ME、m-NAD(P) + -ME、m-NADP+- ME。人類粒線體蘋果酸酶能被反丁烯二酸活化但細胞質蘋果酸酶卻不會,透過胺基酸序列比對,我們將位於調控區上的胺基酸突變到細胞質蘋果酸酶相對應的位置,試圖創造出能被反丁烯二酸調控的人類細胞質蘋果酸酶。此外,我們也解析出人類細胞質蘋果酸酶的結構,希望透過兩個酵素晶體結構的比較,找出人類細胞質蘋果酸酶無法受反丁烯二酸調控的關鍵。人類細胞質蘋果酸酶在一般細胞與腫瘤細胞的表現量與活性無差異;但粒線體蘋果酸酶(m-NAD(P) + -dependent ME)卻會在腫瘤細胞中大量表現。若能找到有效抑制人類粒線體蘋果酸酶卻不影響細胞質蘋果酸酶活性的物質,便能阻斷腫瘤細胞能量來源,且不影響正常細胞能量代謝。在225個植物萃取物中,發現compound A與compound B能選擇性抑制人類粒線體蘋果酸酶。我們將人類粒線體蘋果酸酶結構上具代表性的位置進行點突變,挑選了下列突變株,雙聚體界面上的Q51A/E90A;四聚體界面上的H142A/D568A;exo-site上的R197E與R542V;以及異位調節區上的E59N、R67A、R91A、R67A/R91A、K57S/E59N/ K73E/D102S。將突變株分別加入這兩個化合物進行活性分析,藉此探討化合物可能與人類粒線體蘋果酸酶作用的位置。除異位調節區的突變株外,其他突變株被抑制的效果均與野生型無顯著差異。故推測compound A與compound B可能藉由干擾異位調節區進而抑制酵素活性。透過酵素活性分析,compound A與compound B皆屬於非競爭型抑制劑。由於這兩個藥物能選擇性阻斷腫瘤細胞能量的生成,因此我們認為這兩個化合物相當具有作為抗癌藥物的潛力。
Malic enzymes (ME) are widely distributed in nature, their amino acid sequences are highly conserved across organisms. ME requires divalent cations (Mg2+ or Mn2+) to catalyzes the oxidative decarboxylation of L-malate to pyruvate with the concomitant production of the cofactor NAD(P)H. In mammals, three isoforms of ME have been identified — c-NADP-ME, m-NADP-ME, and m-NAD(P)-ME. Previous studies have indicated that fumarate is bound to R67 and R91 in the allosteric pocket. However, these amino acid residues are conserved in other enzymes that are not activated by fumarate, suggesting that there may be additional factors controlling the activation mechanism. Site-directed mutagenesis was used to replace the fumarate binding pocket, and we tried to create a recombinant human c-NADP-ME with allostiric regulation property by fumarate. Here we report the native crystal structure at 2.5 A resolution of human c-NADP-ME. We hope to find out the critical factor which determines the difference of the allostiric regulation property between human c-NADP-ME and m-NAD(P)-ME. In mammals, the m-NAD(P)+-ME is considered to have an important role in the metabolism of glutamine in rapidly proliferating tissues, especially in tumor cells. Thus, the enzyme could be an ideal target for the rational drug design with a potential application in anticancer therapy. In this study, we found out that compound A and compound B can inhibit human m-NAD(P)-ME efficiently, but not c-NADP-ME. We select mutants, including Q51A/E90A in dimer interface; H142A/D568A in tetramer interface; R197E and R542V in exo-site;E59N、R67A、R91A、R67A/R91A、K57S/E59N/K73E/D102S in allosteric site, to investigate the chemicals probably interacting with which regions of human m-NAD(P)-ME. Only the mutants in allostiric site are poorly suppressed by the chemicals. Therefore, we suggest that compound A and compound B can efficiently inhibit human m-NAD(P)-ME by interfering the allosteric site. Besides, we determine that these two chemicals are noncompetitive inhibitors. Compound A and compound B can specifically inhibit m-NAD(P)-ME, we suggest that these two chemicals may block the energy source of cancer cells and they have potential to be useful anti-cancer drugs.
URI: http://hdl.handle.net/11455/23743
其他識別: U0005-1908201113333000
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