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標題: 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
Determination of the differences in the allosteric site of the human c-NADP-ME and m-NAD(P)-ME
作者: 楊佰寯
Yang, Pai-Chun
關鍵字: malic enzyme;蘋果酸酶結構異構物;analogues
出版社: 生命科學系所
引用: 1. Baggetto LG: Deviant energetic metabolism of glycolytic cancer cells. Biochimie 1992, 74(11):959-974. 2. 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. 3. 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. 4. Chang GG, Huang TM, Chang TC: Reversable dissociation of the catalyically active subunits of pigeon liver the replication of the malic enzyme. Journal of Biochemistry 1988, 254:123-130. 5. Chang GG, Tong L: Structure and function of malic enzymes, a new class of oxidative decarboxylases. Biochemistry 2003, 42(44):12721-12733. 6. 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. 7. Frenkel R: Allosteric characteristics of bovine heart mitochondrial malic enzyme. Biochem Biophys Res Commun 1972, 47(4):931-937. 8. Frenkel R: Regulation and physiological functions of malic enzymes. Current topics in cellular regulation 1975, 9:157-181. 9. Hung HC, Chien YC, Hsieh JY, Chang GG, Liu GY: Functional roles of ATP-binding residues in the catalytic site of human mitochondrial NAD(P)+-dependent malic enzyme. Biochemistry 2005, 44:12737-12745. 10. 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. 11. Hsieh JY, Hung HC: Engineering of the cofactor specificities and isoform-specific inhibition of malic enzyme. J Biol Chem 2009, 284(7):4536-4544. 12. Hsieh JY, Liu JH, Fang YW, Hung HC: Dual roles of Lys57 at the dimer interface of human mitochondrial NAD(P)+-dependent malic enzyme. Biochem. J. 2009, 420:201-209. 13. Hsu RY: Pigeon liver malic enzyme. Mol Cell Biochem 1982, 43(1):3-26. 14. 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. 15. 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. 16. 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. 17. Kuo LS, Chang KY, Hung HC: Hsieh JY, Liu JH, Fang YW, Hung HC: Dual roles of Lys57 at the dimer interface of human mitochondrial NAD(P)+-dependent malic enzyme. Biochem. J. 2009, 420: 201-209. Bioorganic & medicinal chemistry 2009, 17:5414-5419. 18. 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. 19. Landsperger WJ, Harris BG: NAD+-malic enzyme. Regulatory properties of the enzyme from Ascaris suum. J Biol Chem 1976, 251(12):3599-3602. 20. Loeber G, Dworkin MB, Infante A, Ahorn H: Characterization of cytosolic malic enzyme in human tumor cells. FEBS Lett 1994, 344(2-3):181-186. 21. Loeber G, Infante AA, Maurer-Fogy I, Krystek E, Dworkin MB: Human NAD(+)-dependent mitochondrial malic enzyme. cDNA cloning, primary structure, and expression in Escherichia coli. J Biol Chem 1991, 266(5):3016-3021. 22. Mallick S, Harris BG, Cook PF: Kinetic mechanism of NAD:malic enzyme from Ascaris suum in the direction of reductive carboxylation. J Biol Chem 1991, 266:2732-2738. 23. 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. 24. 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. 25. 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 1984a, 259(10):6215-6221. 26. Moreadith RW, Lehninger AL: Purification, kinetic behavior, and regulation of NAD(P)+ malic enzyme of tumor mitochondria. The Journal of biological chemistry 1984b, 259(10):6222-6227. 27. 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. 28. Schimerlik MI, Cleland WW: pH variation of the kinetic parameters and the catalytic mechanism of malic enzyme. Biochemistry 1977, 16(4):576-583. 29. Su KL, Chang KY, Hung HC: Effects of structural analogues of the substrate and allosteric regulator of the human mitochondrial NAD(P)+-dependent malic enzyme. Bioorganic & Medicinal Chemistry 2009, 17:5414-5419. 30. 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. 31. 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. 32. 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):R877-889. 33. Yang Z, Batra R, Floyd DL, Hung HC, Chang GG, Tong L: Potent and competitive inhibition of malic enzymes by lanthanide ions. Biochemical and biophysical research communications 2000a, 274:440-444. 34. Yang Z, Floyd DL, Loeber G, Tong L: Structure of a closed form of human malic enzyme and implications for catalytic mechanism. Nat Struct Biol 2000, 7(3):251-257. 35. 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.
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.
其他識別: U0005-1007201021164300
Appears in Collections:生命科學系所

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