Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23731
標題: 人類鳥胺酸脫羧酶受質專一性之探討
Conversion of substrate specificity of human ornithine decarboxylase
作者: 鄭旭喬
Cheng, Hsu-Chiao
關鍵字: ornithine decarboxylase
鳥胺酸脫羧酶
出版社: 生命科學系所
引用: Almrud JJ, Oliveira MA, Kern AD, Grishin NV, Phillips MA, Hackert ML (2000) Crystal structure of human ornithine decarboxylase at 2.1Å resolution: structure insight to antizyme binding. J Mol Biol 295: 7-16 Auvinen M, Paasinen A, Andersson LC, Hölttä E (1992) Ornithine decarboxylase activity is critical for cell transformation. Nature 360: 355-358 Balbo PB, Patel CN, Sell KG, Adcock RS, Neelakantan S, Crooks PA, Oliveira MA (2003) Spectrophotometric and steady-state kinetic analysis of the biosynthetic arginine decarboxylase of Yersinia pestis utilizing arginine analogues as inhibitors and alternative substrates. Biochemistry 42: 15189-96 Coffino P (2001) Antizyme, a mediator of ubiquitin-independent proteasomal degradation. Biochimie 83: 319-323 Grishin NV, Osterman AL, Brooks HB, Phillips MA, Goldsmith EJ (1999) X-ray structure of ornithine decarboxylase from Trypanosoma brucei: the native structure and the structure in complex with alpha-difluoromethylornithine. Biochemistry 38: 15174-84. Halaris A, Plietz J (2007) Agmatine : metabolic pathway and spectrum of activity in brain. CNS Drugs 21: 85-900. Jackson LK, Brooks HB, Osterman AL, Goldsmith EJ, Phillips MA (2000) Altering the reaction specificity of eukaryotic ornithine decarboxylase. Biochemistry 39: 11247-11257 Jackson LK, Brooks HB, Myers DP, Phillips MA (2003) Ornithine decarboxylase promotes catalysis by binding the carboxylate in a buried pocket containing phenylalanine 397. Biochemistry 42: 2933-40 Keren-Paz A, Bercovich Z, Porat Z, Erez O, Brener O, Kahana C (2006) Overexpression of antizyme-inhibitor in NIH3T3 fibroblasts provides growth advantage through neutralization of antizyme functions. Oncogene 25: 5163-5172 Kern AD, Oliveira MA, Coffino P, Hackert ML (1999) Structure of mammalian ornithine decarboxylase at 1.6Å resolution: stereochemical implications of PLP-dependent amino acid decarboxylsae. Structure 7: 567-581 Lee YS, Cho YD (2001) Identification of essential active-site residues in ornithine decarboxylase of Nicotiana glutinosa decarboxylating both L-ornithine and L-lysine. Biochem J 360: 657-65 Li G, Regunathan S, Barrow CJ, Eshraghi J, Cooper R, Reis DJ (1994) Agmatine: an endogenous clonidine-displacing substance in the brain. Science 263: 966-9 López-Contreras AJ, López-Garcia C, Jiménez-Cervantes C, Cremades A, Peñafiel R (2006) Mouse ornithine decarboxylase-like gene encodes an antizyme inhibitor devoid of ornithine and arginine decarboxylating activity. J Biol Chem 281: 30896-906. Marcora MS, Cejas S, González NS, Carrillo C, Algranati ID (2010) Polyamine biosynthesis in Phytomonas: biochemical characterisation of a very unstable ornithine decarboxylase. Int J Parasitol 40: 1389-94 Myers DP , Jackson LK , Ipe VG, Murphy GE, Phillips MA(2001) Long-range interactions in the dimer interface of ornithine decarboxylase are important for enzyme function. Biochemistry 40: 13230-6 Osterman AL, Kinch LN, Grishin NV, Phillips MA (1995) Acidic residues important for substrate binding and cofactor reactivity in eukaryotic ornithine decarboxylase identified by alanine scanning mutagenesis. J Biol Chem 270: 11797-802. Pitkänen LT, Heiskala M, Andersson LC (2001) Expression of a novel human ornithine decarboxylase-like protein in the central nervous system and testes. Biochem Biophys Res Commun 287: 1051-7 Pegg AE (2006) Regulation of ornithine decarboxylase. J Biol Chem 281: 14529-14532 Reis DJ, Regunathan S (1998) Agmatine: a novel neurotransmitter? Adv Pharmacol 42: 645-9. Regunathan S, Youngson C, Raasch W, Wang H, Reis DJ. (1996) Imidazoline receptors and agmatine in blood vessels: a novel system inhibiting vascular smooth muscle proliferation. J Pharmacol Exp Ther 276: 1272-82 Regunathan S, Feinstein DL, Reis DJ (1999) Anti-proliferative and anti-inflammatory actions of imidazoline agents. Are imidazoline receptors involved? Ann N Y Acad Sci 881: 410-9 Satriano J, Matsufuji S, Murakami Y, Lortie MJ, Schwartz D, Kelly CJ, Hayashi S, Blantz RC (1998) Agmatine suppresses proliferation by frameshift induction of antizyme and attenuation of cellular polyamine levels. J Biol Chem 273: 5313-6. Schaeffer JM, Donatelli MR (1990) Characterization of a high-affinity membrane-associated ornithine decarboxylase from the free-living nematode Caenorhabditis elegans. Biochem J 270: 599-604 Shantz LM, Coleman CS, Pegg AE (1996) Expression of an ornithine decarboxylase dominant-negative mutant reverses eukaryotic initiation factor 4E-induced cell transformation. Cancer Res 56: 5136-5140 Zhu MY, Iyo A, Piletz JE, Regunathan S. (2004) Expression of human arginine decarboxylase, the biosynthetic enzyme for agmatine. Biochim Biophys Acta 1670: 156-64.
摘要: 人類鳥胺酸脫羧酶(ornithine decarboxylase, ODC; EC 4. 1. 1. 17)為體內多元胺 (polyamines) 合成過程中參與第一步驟及速率決定的酵素,其功能為催化鳥胺酸 (L-ornithine) 形成腐胺 (putrescine) 與二氧化碳。具有酵素催化活性的鳥胺酸脫羧酶是以雙聚體的形式存在。而精胺酸脫羧酶 (arginine decarboxylase , ADC; EC 4.1.1.19) 能催化精胺酸 (L-arginine) 形成胍戊胺 (agmatine) 與二氧化碳,胍戊胺再經由agmatinase (EC 3.5.3.11) 催化則能進一步形成腐胺。人類鳥胺酸脫羧酶與精胺酸脫羧酶胺基酸序列有很高的相似度,鳥胺酸脫羧酶的主要受質為鳥胺酸,但其對精胺酸仍有些微活性,而精胺酸脫羧酶之受質則為精胺酸。我們的研究目標是去找出決定鳥胺酸脫羧酶與精胺酸脫羧酶受質專一性的胺基酸位置。經由鳥胺酸脫羧酶與精胺酸脫羧酶的序列比對結果,找出活性中心或活性中心附近不保留胺基酸位置,將鳥胺酸脫羧酶序列 (D38、D88、T93、R154、R165、Y331及C360) 改成精胺酸脫羧酶序列(A38、S88、A93、C154、C165、F331及V360)。這些突變型利用酵素動力學實驗來分析其Km 及kcat 值,並與野生型鳥胺酸脫羧酶比較。實驗結果發現D88S、R154C、C360V與野生型ODC有顯著差異。對受質鳥胺酸的Km 值明顯上升,kcat 值則明顯下降。當受質換成精胺酸時,此三個突變型對精胺酸的kcat 值明顯提升。之後再分別去觀察雙重突變型 (ODC-D88S/R154C、ODC-D88S/C360V及ODC-R154C/C360V) 及三重突變型 (ODC-D88S/R154C/C360V) ODC的結果。發現ODC-D88S/R154C、ODC-D88S/C360V及ODC-R154C/C360V對鳥胺酸的Km 值與單一突變型ODC差異不大,但其對鳥胺酸的kcat 值則更低,表示其對鳥胺酸的催化能力愈低。在受質為精胺酸方面,此三種突變型對受質的EC50值與野生型或單一突變型比較均無明顯差異,但kcat 值則各比單一突變型高約1.3~2.2倍。ODC-D88S/R154C/C360V對鳥胺酸的kcat 值及對精胺酸的EC50與kcat 值雖與野生型ODC有明顯差異,但與三個雙重突變型比則沒有更顯著的差異。對鳥胺酸Km 值的部分,其Km 值高於野生型ODC 13倍,雙重突變型ODC 2~8倍,顯示其對受質鳥胺酸的活性更低。我們的研究結果發現胺基酸88、154及360是影響人類鳥胺酸脫羧酶與精胺酸脫羧酶受質專一性的關鍵位置。
The human ornithine decarboxylase (ODC; EC 4. 1. 1. 17), which is the rate-limiting to polyamines synthesis, catalyzes the decarboxylation of L-ornithine to form putrescine and carbon dioxide. The active form of ODC is obligate homodimer. The arginine decarboxylase (ADC; EC 4.1.1.19) is a key enzyme involved in the biosynthesis of putrescine. ADC catalyzes the decarboxylation of L-arginine to form agmatine and carbon dioxide, and agmatine is catabolised to form putrescine by agmatinase. Base on the multiple sequence alignments of ODC and ADC, the non-conserved amino acid residues in the active site of ODC (D38, D88, T93, R154, R165, Y331 and C360) were changed to the amino acid residues of ADC (A38, S88, A93, C154, C165, F331 and V360). When we utilized ornithine as the substrate, the Km values for D88S、R154C and C360V were significantly elevated,and kcat values were reduced after enzyme kinetic assay. Then we tested double (ODC-D88S/R154C、ODC-D88S/C360V and ODC-R154C/C360V) and triple (ODC-D88S/R154C/C360V) mutans with the same assay. The results indicate that the residues 88, 154 and360 were important for substrate specificity of human ornithine decarboxylase and arginine decarboxylase.
URI: http://hdl.handle.net/11455/23731
其他識別: U0005-1808201117543000
Appears in Collections:生命科學系所

文件中的檔案:

取得全文請前往華藝線上圖書館



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.