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標題: 探討四級結構組成與酵素催化之關係:人類粒腺體蘋果酸酶和細胞質蘋果酸酶的差異
Study on the relationship between the quaternary structure organizations and enzyme catalysis : Differences between m-NAD(P)-ME and c-NADP-ME
作者: 陳紹宏
Chen, Shao-Hung
關鍵字: malic enzyme
出版社: 生命科學系所
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Reitzer LJ, Wice BM, Kennell D: Evidence that glutamine, not sugar, is the major energy source for cultured HeLa cells. The Journal of biological chemistry 1979, 254(8):2669-2676. 13. Teller JK, Fahien LA, Davis JW: Kinetics and regulation of hepatoma mitochondrial NAD(P) malic enzyme. The Journal of biological chemistry 1992, 267(15):10423-10432. 14. Fahien LA, Teller JK: Glutamate-malate metabolism in liver mitochondria. A model constructed on the basis of mitochondrial levels of enzymes, specificity, dissociation constants, and stoichiometry of hetero-enzyme complexes. The Journal of biological chemistry 1992, 267(15):10411-10422. 15. Moreadith RW, Lehninger AL: The pathways of glutamate and glutamine oxidation by tumor cell mitochondria. Role of mitochondrial NAD(P)+-dependent malic enzyme. The Journal of biological chemistry 1984, 259(10):6215-6221. 16. Sauer LA, Dauchy RT, Nagel WO, Morris HP: Mitochondrial malic enzymes. Mitochondrial NAD(P)+-dependent malic enzyme activity and malate-dependent pyruvate formation are progression-linked in Morris hepatomas. The Journal of biological chemistry 1980, 255(9):3844-3848. 17. Chang GG, Tong L: Structure and function of malic enzymes, a new class of oxidative decarboxylases. Biochemistry 2003, 42(44):12721-12733. 18. 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. 19. Yang Z, Floyd DL, Loeber G, Tong L: Structure of a closed form of human malic enzyme and implications for catalytic mechanism. Nature structural biology 2000, 7(3):251-257. 20. Yang Z, Zhang H, Hung HC, Kuo CC, Tsai LC, Yuan HS, Chou WY, Chang GG, Tong L: Structural studies of the pigeon cytosolic NADP(+)-dependent malic enzyme. Protein Sci 2002, 11(2):332-341. 21. Coleman DE, Rao GS, Goldsmith EJ, Cook PF, Harris BG: Crystal structure of the malic enzyme from Ascaris suum complexed with nicotinamide adenine dinucleotide at 2.3 A resolution. Biochemistry 2002, 41(22):6928-6938. 22. 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. 23. Chang GG, Huang TM, Chang TC: Reversible dissociation of the catalytically active subunits of pigeon liver malic enzyme. The Biochemical journal 1988, 254(1):123-130. 24. Chou WY, Huang SM, Chang GG: Functional roles of the N-terminal amino acid residues in the Mn(II)-L-malate binding and subunit interactions of pigeon liver malic enzyme. Protein engineering 1997, 10(10):1205-1211. 25. Chou WY, Liu MY, Huang SM, Chang GG: Involvement of Phe19 in the Mn(2+)-L-malate binding and the subunit interactions of pigeon liver malic enzyme. Biochemistry 1996, 35(30):9873-9879. 26. Chang HC, Chang GG: Involvement of single residue tryptophan 548 in the quaternary structural stability of pigeon cytosolic malic enzyme. The Journal of biological chemistry 2003, 278(26):23996-24002. 27. Chang HC, Chen LY, Lu YH, Li MY, Chen YH, Lin CH, Chang GG: Metal ions stabilize a dimeric molten globule state between the open and closed forms of malic enzyme. Biophysical journal 2007, 93(11):3977-3988. 28. Chang HC, Chou WY, Chang GG: Effect of metal binding on the structural stability of pigeon liver malic enzyme. The Journal of biological chemistry 2002, 277(7):4663-4671.
摘要: 蘋果酸酶催化蘋果酸地氧化脫羧反應,產生丙酮酸和二氧化碳,並伴隨輔酶NAD(P)+還原成NAD(P)H,此反應需要二價金屬離子作為輔因子。在哺乳類體內有三種異構酶: c-NADP-ME、m-NAD(P)-ME和m-NADP-ME。粒腺體蘋果酸酶(m-NAD(P)-ME)對蘋果酸的結合具有協同作用;而反丁烯二酸會增加其酵素活性但會使此協同作用消失。不同物種的蘋果酸酶具有不同的四級結構強度。在中性酸鹼度之下,鴿子的c-NADP-ME是以單純四聚體(沉降係數約為10 S)的形式存在;而人類的m-NAD(P)-ME則是四聚體和二聚體(沉降係數分別為9.5 S和6.5 S)混和的狀態。本實驗的目的在於探討人類兩種蘋果酸酶(c-NADP-ME、m-NAD(P)-ME)在形成四聚體之接觸介面的重要可解離氨基酸,以及四級結構強度對於這兩種異構酶催化作用的影響。我們使用LigPlot和DimPlot找出在四個單元體結合介面可能提供離子對的氨基酸,並用定點突變將其突變為丙胺酸(alanine)以消除氨基酸側鏈的電荷,再以分析型超高速離心機(analytical ultracentrifuge)分析其四級結構狀態,並進行酵素活性測定。實驗結果顯示,四級結構的改變並不影響c-NADP-ME的酵素活性,但m-NAD(P)-ME的活性以及協同作用卻有明顯降低的現象。這表示c-NADP-ME四個單元體的催化中心並不會互相影響,形成四聚體的功能主要是為成整體結構的穩定;而m-NAD(P)-ME的四級結構不論在整個結構穩定或是酵素催化的調節上皆很重要。這結果也反映了這兩種異構酶之調節差異,以及其在生理作用上所扮演的不同角色。
Malic enzymes catalyze the reversible oxidative decarboxylation of L-malate to yield pyruvate and CO2, with the reduction of NAD(P)+ to NAD(P)H. And this reaction needs divalent metal ion (Mg2+ or Mn2+) for enzyme activity. There are three isoforms of malic enzymes in mammals: c-NADP-ME, m-NAD(P)-ME, and m-NADP-ME. m-NAD(P)-ME is a cooperative enzyme with malate binding. Fumarate can increase the activity of m-NAD(P)-ME but abolish the sigmoidal kinetics with respect to malate. The quaternary structures of malic enzymes from different species have various strengths. At neutral pH condition, pigeon c-NADP-ME exists as a single species of tetramer with a sedimentation coefficient of approximately 10 S, whereas human m-NAD-ME exists as a mixture of tetramer and dimer with 9.5 S and 6.5 S, respectively. The sedimentation data of human c-NADP-ME has not been reported. The goal of this research is to treat about the essential ionic residues in the interfaces of human c-NADP-ME and m-NAD(P)-ME, as well as effects of the quaternary structure organization on their catalysis. We used LigPlot and DimPlot to find the possible residues that contribute ionic bonding at the interfaces in these two malic enzyme isoforms and create mutants by site-directed mutagenesis. Then the proteins were analyzed by analytical ultracentrifuge (AUC) for their quaternary structures and assayed for the activities. We found that the change in quaternary structures would not affect the activities of c-NADP-ME but would affect m-NAD(P)-ME in both activities and cooperative behavior. The mutants that caused quaternary structural changes in m-NAD(P)-ME have significantly reduced activities and Hill coefficient (h) compared to WT. This implied that active sites of human c-NADP-ME do not affect each other and the role of polymeric state is to maintain the stability of overall structure, but not for the regulating enzyme activity. But the quaternary structure of m-NAD(P)-ME is important not only to the structural stability but also the regulation in its catalysis.
其他識別: U0005-2107200813353800
Appears in Collections:生命科學系所



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