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Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22713
標題: 利用定點突變方法在人類細胞質蘋果酸酶建立異位調節區和菸鹼醯胺腺嘌呤二核苷酸結合位
Creation of an allosteric site and a NAD+ binding site in cytosolic NADP+-dependent malic enzyme.
作者: 陳孟君
Chen, Meng-Chun
關鍵字: malic enzyme;蘋果酸酶
出版社: 生命科學系所
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Biochem J 1994, 304 ( Pt 3):687-692. 12.Mandella RD, Sauer LA: The mitochondrial malic enzymes. I. Submitochondrial localization and purification and properties of the NAD(P)+-dependent enzyme from adrenal cortex. J Biol Chem 1975, 250(15):5877-5884. 13.Frenkel R: Regulation and physiological functions of malic enzymes. Current topics in cellular regulation 1975, 9:157-181. 14.Goodridge AG, Klautky SA, Fantozzi DA, Baillie RA, Hodnett DW, Chen W, Thurmond DC, Xu G, Roncero C: Nutritional and hormonal regulation of expression of the gene for malic enzyme. Progress in nucleic acid research and molecular biology 1996, 52:89-122. 15.McKeehan WL, McKeehan KA: Changes in NAD(P)+-dependent malic enzyme and malate dehydrogenase activities during fibroblast proliferation. J Cell Physiol 1982, 110(2):142-148. 16.Moreadith RW, Lehninger AL: The pathways of glutamate and glutamine oxidation by tumor cell mitochondria. Role of mitochondrial NAD(P)+-dependent malic enzyme. J Biol Chem 1984, 259(10):6215-6221. 17.Baggetto LG: Deviant energetic metabolism of glycolytic cancer cells. Biochimie 1992, 74(11):959-974. 18.Tsai LC, Kuo CC, Chou WY, Chang GG, Yuan HS: Crystallization and preliminary x-ray diffraction analysis of malic enzyme from pigeon liver. Acta crystallographica 1999, 55(Pt 11):1930-1932. 19.Baker PJ, Thomas DH, Barton CH, Rice DW, Bailey E: Crystallization of an NADP+-dependent malic enzyme from rat liver. Journal of molecular biology 1987, 193(1):233-235. 20.Clancy LL, Rao GS, Finzel BC, Muchmore SW, Holland DR, Watenpaugh KD, Krishnamurthy HM, Sweet RM, Cook PF, Harris BG: Crystallization of the NAD-dependent malic enzyme from the parasitic nematode Ascaris suum. Journal of molecular biology 1992, 226(2):565-569. 21.Bhargava G, Mui S, Pav S, Wu H, Loeber G, Tong L: Preliminary crystallographic studies of human mitochondrial NAD(P)(+)-dependent malic enzyme. J Struct Biol 1999, 127(1):72-75. 22.Xu Y, Bhargava G, Wu H, Loeber G, Tong L: Crystal structure of human mitochondrial NAD(P)(+)-dependent malic enzyme: a new class of oxidative decarboxylases. Structure 1999, 7(8):877-889. 23.Yang Z, Lanks CW, Tong L: Molecular mechanism for the regulation of human mitochondrial NAD(P)+-dependent malic enzyme by ATP and fumarate. Structure 2002, 10(7):951-960. 24.Tao X, Yang Z, Tong L: Crystal structures of substrate complexes of malic enzyme and insights into the catalytic mechanism. Structure 2003, 11(9):1141-1150. 25.Schimerlik MI, Cleland WW: pH variation of the kinetic parameters and the catalytic mechanism of malic enzyme. Biochemistry 1977, 16(4):576-583. 26.Kiick DM, Harris BG, Cook PF: Protonation mechanism and location of rate-determining steps for the Ascaris suum nicotinamide adenine dinucleotide-malic enzyme reaction from isotope effects and pH studies. Biochemistry 1986, 25(1):227-236. 27.Sauer LA: Mitochondrial NAD-dependent malic enzyme: a new regulatory enzyme. FEBS Lett 1973, 33(2):251-255. 28.Sauer LA: An NAD- and NADP-dependent malic enzyme with regulatory properties in rat liver and adrenal cortex mitochondrial fractions. Biochem Biophys Res Commun 1973, 50(2):524-531. 29.Karsten WE, Pais JE, Rao GS, Harris BG, Cook PF: Ascaris suum NAD-malic enzyme is activated by L-malate and fumarate binding to separate allosteric sites. Biochemistry 2003, 42(32):9712-9721. 30.Frenkel R: Allosteric characteristics of bovine heart mitochondrial malic enzyme. Biochem Biophys Res Commun 1972, 47(4):931-937. 31.Hsu WC, Hung HC, Tong L, Chang GG: Dual functional roles of ATP in the human mitochondrial malic enzyme. Biochemistry 2004, 43(23):7382-7390. 32.Landsperger WJ, Harris BG: NAD+-malic enzyme. Regulatory properties of the enzyme from Ascaris suum. J Biol Chem 1976, 251(12):3599-3602. 33.Lai CJ, Harris BG, Cook PF: Mechanism of activation of the NAD-malic enzyme from Ascaris suum by fumarate. Arch Biochem Biophys 1992, 299(2):214-219. 34.Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry 1976, 72:248-254. 35.Brown PH, Schuck P: Macromolecular size-and-shape distributions by sedimentation velocity analytical ultracentrifugation. Biophys J 2006, 90(12):4651-4661. 36.Hung HC, Kuo MW, Chang GG, Liu GY: Characterization of the functional role of allosteric site residue Asp102 in the regulatory mechanism of human mitochondrial NAD(P)+-dependent malate dehydrogenase (malic enzyme). Biochem J 2005, 392(Pt 1):39-45. 37.Kuo CC, Tsai LC, Chin TY, Chang GG, Chou WY: Lysine residues 162 and 340 are involved in the catalysis and coenzyme binding of NADP(+)-dependent malic enzyme from pigeon. Biochem Biophys Res Commun 2000, 270(3):821-825.
摘要: 
人類細胞質蘋果酸酶 (c-NADP-ME) 在有二價金屬離子 (如錳、鎂離子) 的存在下，能將蘋果酸及 NADP+催化形成丙酮酸、二氧化碳及NADPH，進行可逆的氧化脫羧反應。人類細胞質蘋果酸酶在結構上是由四個完全相同的單體組合而成的四聚體，可分為結合較緊密的雙聚體界面和較鬆散的四聚體界面，每個單體都具有各自的活化中心。不同於c-NADP-ME，人類粒線體蘋果酸酶 (c-NAD-ME)在雙聚體界面處有一異位調節區，可與反丁烯二酸結合，促使活性提升；而腺苷三磷酸則會和輔因子競爭結合到活化中心，抑制活性。在本篇研究中，我們藉由胺基酸序列比對分析，挑選第57、59、73、74、78、80、87和102號胺基酸，進行定點突變，希望在c-NADP-ME建立一個異位調節區。從實驗結果得知突變型S57K/N59E/E73K/S102D具有最好的活化效果，指出置換Ser57、Asn59、Glu73、和Ser102這四個胺基酸有助於異位調節區建立。另外，先前的研究發現第346、347、362號胺基酸可能參與蘋果酸酶輔因子NADP+的選擇。我們製造三個單點突變 (S346K, K347Y, K362Q)，三個雙突變 (S346K/K347Y, S346K/K362Q, K347Y/K362Q)和一個三突變 (S346K/K347Y/K362Q) 進一步探討這三個胺基酸對於輔因子選擇性的影響。從酶動力學的結果得知，相較於野生型，突變型 S346K/K347Y/K362Q的Km(NADP) 值增加4822倍而 Km(NAD) 值降低了2倍，另外，其kcat(NAD)值也提升至接近野生型的kcat(NADP)值。顯示S346K/K347Y/K362Q成功地將c-NADP-ME的輔因子從NADP+轉換成 NAD+。除此之外，我們也進一步探討活化中心的突變對於腺苷三磷酸所造成的抑制現象是否有所影響。實驗結果指出無論以NADP+或NAD+作為輔因子，活化中心電荷的平衡與否影響了腺苷三磷酸對人類細胞質蘋果酸酶活性抑制的強弱。

Cytosolic NADP+-dependent malic enzyme (c-NADP-ME) catalyzes a reversible oxidative decarboxylation converting L-malate into CO2 and pyruvate, concomitant with reduction of NADP+ to NADPH. Divalent metal ion (Mn2+ or Mg2+) is required for catalysis. According to the previous studies, mitochondrial malic enzyme (m-NAD-ME) is considered as an allosteric enzyme with fumarate as an activator and ATP as an inhibitor. In this study, we created a series of multiple mutations based on the sequence alignments to build a fumarate binding site for human c-NADP-ME. The optimal activating effect by fumarate was observed in the quadruple mutant (S57K/N59E/E73K/S102D). Our kinetic data clearly indicate that substitution of Ser57, Asn59, Glu73, and Ser102 is helpful in creation of fumarate binding site. In addition, previous kinetic studies have suggested that Ser346, Lys347, and Lys362 might be responsible for NADP+ selectivity. We further delineated the relationships of these three residues for the cofactor selectivity in human c-NADP-ME by creating three single mutants (S346K, K347Y, K362Q), three double mutants (S346K/K347Y, S346K/K362Q, K347Y/ K362Q), and one triple mutant (S346K/K347Y/K362Q). The cofactor preference shift from NADP+ to NAD+ was accomplished with the triple mutant, owing to the Km(NADP) and Km(NAD) values of the triple mutant were increased by 4822-fold and decreased by about 2-fold, respectively, compared with those of wild-type enzyme. Besides, the kcat(NAD) of triple mutant enzyme increased to a similar level of kcat(NADP) of wild-type enzyme. Thus, based on our kinetic data, we suggested that the balance of electric charge of these mutant enzymes might result in differential ATP inhibition by utilizing NAD+ or NADP+ as cofactor.
URI: http://hdl.handle.net/11455/22713
其他識別: U0005-2007200814005500
Appears in Collections:生命科學系所

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