黃嘌呤氧化酶與受質6-formylpterin作用機制及其中間產物形式之研究

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研究生：	陳宥蓁 Yu-Chen Chen
論文名稱：	黃嘌呤氧化酶與受質6-formylpterin作用機制及其中間產物形式之研究 Investigation of interaction mechanism and intermediate complex between xanthine oxidase and 6-formylpterin
指導教授：	黃國柱 Kuo-Chu Hwang
口試委員:
學位類別：	碩士 Master
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2007
畢業學年度：	95
語文別：	中文
論文頁數：	122
中文關鍵詞：	黃嘌呤氧化酶、同位素效應、交互作用、席夫鹼
外文關鍵詞：	Xanthine oxidase, XOD, Isotope effect, cooperativity, Schiff base
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黃嘌呤氧化酶( xanthine oxidase ; XOD ) ，分子量約為290kDa，為一同相二聚體(homodimer)，為代謝黃嘌呤(Xanhine)與亞黃嘌呤(hypoxanthine)成為尿酸的主要酵素，而尿酸過高易引起痛風及並其併發症狀，目前以異位次黃嘌呤(allopurinol)為黃嘌呤氧化酶之良好抑制劑而作為治療痛風的主要藥物。研究黃嘌呤氧化酶之反應動力學與催化作用，有助於更精確的掌握投藥量與開發更有效之藥物。
早期研究認為黃嘌呤氧化酶兩個單體間之催化行為是各自獨立，不受彼此影響。本研究利用不同活性的黃嘌呤氧化酶(AA、AI、II form)觀察其對受質6-formylpterin(6FP)與氘化之6-formylpterin (D-6FP)的催化行為。實驗中發現，當黃嘌呤氧化酶兩個單體其中之一佔有受質時，會造成另一個單體催化能力的改變，期間存在了顯著的交互作用。
此外，黃嘌呤氧化酶催化6FP與D-6FP時，其同位素效應並不明顯；而在照光的條件下，同位素效應相繼提升，進而推斷黃嘌呤氧化酶催化6FP的過程中，6FP的酮基與很有可能與XOD氨基酸序列上的離胺酸(lysine)或精胺酸(arginine)形成具有席夫鹼(Schiff base)結構的中間產物，而產生了”光誘發催化”，藉由蛋白質水解技術，觀測到6FP與黃嘌呤氧化酶之氨基酸序列產生了化學鍵結，合理解釋了為何在催化受質6FP無法看到顯著的同位素效應之故。

摘要.............................................................I
目錄...........................................................III
圖目錄..........................................................VI
表目錄.........................................................XII
第一章  前言.....................................................1
第二章  文獻回顧.................................................3
2-1  常用的酵素探力學探討模型----Michaelis-Menten模型........... 3
2-2  酵素活性調控(Regulation of enzymes)............    .........7
2-3  由異位性質(allostery)觀察交互作用(cooperativity)現象.......11
2-4  黃嘌呤氧化酶( xanthine oxidase ; XOD ).....................14
2-4-1  黃嘌呤氧化酶結構.........................................14
2-4-2  黃嘌呤氧化酶催化與輔酶關係...............................16
2-4-3  影響黃嘌呤氧化酶活性有無的關鍵...........................18
2-4-4  黃嘌呤氧化酶的催化機制.................................  23
2-5  黃嘌呤氧化酶相關疾病.......................................25
2-5-1  痛風（gout）.............................................25
2-5-2  缺血再灌流傷害（Ischemia-reperfusion injury）............28
2-6  受質濃度對於黃嘌呤氧化酶的影響.............................30
2-7  黃嘌呤氧化酶的受質.........................................32
2-7-1  黃嘌呤氧化酶的常見受質...................................32
2-7-2  6-Formylpterin (6FP).....................................33
第三章  實驗原理與方法..........................................35
3-1  實驗藥品和儀器.............................................35
3-1-1  實驗藥品.................................................35
3-1-2  實驗儀器.................................................37
3-2  XOD純化原理................................................38
3-2-1 黃嘌呤氧化酶純化原理......................................38
3-2-2 葡萄糖氧化酶除氧原理......................................41
3-3  XOD純化方法................................................42
3-3-1  製備親合性管柱 ( EAH-Sepharose™4B gel )..................42
3-3-2  黃嘌呤氧化酶(Xanthine Oxidase，簡稱XOD)純化步驟..........43
3-4  黃嘌呤氧化酶濃度與活性測定.................................45
3-4-1  黃嘌呤氧化酶濃度測定.....................................45
3-4-2  黃嘌呤氧化酶活性測定.....................................46
3-5  黃嘌呤氧化酶氨基酸序列.....................................47
3-6  受質6-formylpterin (6FP)...................................49
3-6-1  6-formylpterin簡介.......................................49
3-6-2  6FP製備方法..............................................50
3-7  XOD-6FP複合物製備與蛋白質水解..............................51
3-7-1  XOD-6FP複合物製備與蛋白質水解原理........................51
3-7-2  XOD-6FP複合物製備與蛋白質水解方法........................52
第四章  實驗結果與討論..........................................53
4-1  XOD純化與6FP...............................................53
4-1-1  XOD純化結果..............................................53
4-1-2  XOD之AA、AI、II form純化結果.............................54
4-2  受質6FP與D-6FP對於黃嘌呤氧化酶之交互作用影響...............57
4-2-1  6FP對於AA from黃嘌呤氧化酶之交互作用影響.................58
4-2-1a  k2、k3值的測量..........................................58
4-2-1b  k3’值的測量............................................61
4-2-2  D-6FP對於AA from黃嘌呤氧化酶之交互作用影響...............65
4-2-2a  k2、k3值的測量..........................................65
4-2-2b  k3’值的測量............................................67
4-2-3  6FP對於AI from黃嘌呤氧化酶之交互作用影響.................70
4-2-3a  k2、k3值的測量..........................................70
4-2-3b  k3’值的測量............................................73
4-2-4  D-6FP對於AI from黃嘌呤氧化酶之交互作用影響...............77
4-2-4a  k2、k3值的測量..........................................77
4-2-4b  k3’值的測量............................................79
4-2-5  6FP對於II from黃嘌呤氧化酶之交互作用影響.................83
4-2-5a  k2、k3值的測量..........................................83
4-2-5b  k3’值的測量............................................85
4-2-6  D-6FP對於II from黃嘌呤氧化酶之交互作用影響...............87
4-2-6a  k2、k3值的測量..........................................87
4-2-6b  k3’值的測量............................................88
4-2-7  受質6FP與D-6FP對於XOD之交互作用影響之比較................90
4-3  照光對於XOD-6FP複合物的影響................................94
4-3-1  照光波長的選用...........................................94
4-3-2  XOD-6FP與XOD-D6FP複合物照光反應..........................95
4-4  利用蛋白質水解法分析XOD-6FP的細部鍵結......................99
參考文獻.......................................................109
附件1..........................................................115
附件2..........................................................120

                                

1. Hille, R., and Nishino, T. (1995). "Flavoprotein structure and mechanism. 4. Xanthine oxidase and xanthine dehydrogenase."Faseb. J. 9(11): 995-1003.
2. Hille, R. (1996). "The Monomuclear Molybdenum Enzymes." Chem. Rev. 96(7): 2757-816.
3. Hille, R. (2005). "Molybdenum-containing hydroxylases." Arch. Biochem. Biophys. 433(1):107-11
4. Enroth, C., Eger, B. T., et al. (2000). "Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: Structure-based mechanism of conversion." Proc. Natl. Acad. Sci. U.S.A. 97(20): 10723-8.
5. Massey, V., Brumby, P. E., et al. (1969). "Studies on milk xanthine oxidase. Some spectral and kinetic properties." J. Biol. Chem. 244(7): 1682-91.
6. Hart, L. I., McGartoll, M. A., et al. (1970). "The composition of milk xanthine oxidase." Biochem. J. 116(5): 851-64.
7. Thijssen, H. H. (1973). "A simple method for preparing 2-amino-4-hydroxy-6-formylpteridine, a precursor of the pteridine substrate of dihydropteroate biosynthesis." Anal. Biochem. 54(2): 609-11.
8. Nishino, T., Nishino, T., et al. (1981). "Purification of highly active milk xanthine oxidase by affinity chromatography on Sepharose 4B/folate gel." Febs. Lett. 131(2): 369-72
9. Nishino, T., and Tsushima, K. (1986). "Interaction of milk xanthine oxidase with folic acid. Inhibition of milk xanthine oxidase by folic acid and separation of the enzyme into tow fractions on Sepharose 4B/ folate gel." J. Biol. Chem. 261(24): 11242-6
10. Massey, V., and Edmondson D. (1970). "On the mechanism of inactivation of xanthine oxidase by cyanide." J. Biol. Chem. 245(24): 6595-8.
11. Massey, V., Brumby, P. E., et al. (1969). "Studies on mil xanthine oxidase. Some spectral and kinetic properties." J. Biol. Chem. 244(7): 1682-91.
12. Massey, V., Komai, H., et al. (1970). "On the mechanism of inactivation of xanthine oxidase by allopurinol and other pyrazolo [3,4-d]pyrimidines." J. Biol. Chem. 245(11): 2837-44.
13. Edmondson, D., Massey, V., et al. (1972). "The resolution of active and inactive xanthine oxidase by affinity chromatography." J. Biol. Chem. 247(5): 1597-604.
14. http://en.wikipedia.org/wiki/Ischemia
http://en.wikipedia.org/wiki/Reperfusion_injury
15. http://en.wikipedia.org/wiki/gout
16. Patrick , L., “Medicinal Chemistry”, Third ed. Chap. 4, p59~61.
17. Patrick, L., “Medicinal Chemistry”, Third ed. Chap. 4
18. Stryer , L., “Biochemistry”, Fourth ed. Chap. 10
19. David, L. L., and Michael, M. M. “Principles of Biochemistry”, Fourth ed. Chap. 5
20. Howes, B. D., Bray, R. C., et al. (1996). "Evidence favoring molybdenum-carbon bond formation in xanthine oxidase action: 17O- and 13C-ENDOR and kinetic studies." Biochemisty 35(5): 1432-43.
21. Skibo, E. B., Gilchrist, J. H., et al. (1987). "Eloctronic probes of the mechanism of substrate oxidation by buttermilk xanthine oxidase: role of the active-site nucleophile in oxidation." Biochemisty 26(11): 3032-7.
22. Xia, M., Dempski, R., et al. (1999). "The reductive half-reaction of xanthine oxidase. Reaction with aldehyde substrates and identification of the catalytically labile oxygen." J. Biol. Chem. 274(6): 3323-30.
23. Coughlan, M. P., Rajagopalan, K. V., et al. (1969). "The role of molybdenum in xanthine oxidase and related enzymes. Reactivity with cyanide, aresenite, and methanol" J. Biol. Chem. 244(10): 2658-63.
24. Palmber, G., Bray, R. C., et al. (1964). "Direct studies on the electrion transfer sequence in xanthine oxidase by electron paramagnetic resonance spectroscopy. Ι. Techniques and description of spectra. " J. Biol. Chem. 239: 2657-66.
25. Wu, C. L.,and Melton, D. W. (1993). "Production of a model for Lesch-Nyhan syndrome in hypoxanthine phosphoribosyltransferase-deficient mice. " Nat. Genet. 3(3): 235-40.
26. http://www.benbest.com/cryonics/ischemia.html
27. http://en.wikipedia.org/wiki/Reperfusion_injury
28. Hofstee, B. H. (1955). "On the mechanism of inhibition of xanthine oxidase by the substrate xanthine " J. Biol. Chem. 216(1): 235-44.
29. Priest, D. G., and Fisher, J. R. (1969). "Substrate activation with a xanthine oxidase reaction." Eur. J. Biochem. 10(3): 439-44.
30. Hille, R., George, G. N., et al. (1989). "EXAFS analysis of xanthine oxidase complexes with alloxanthine, violapterin, and 6-pteridylaldehyde." Inorg. Chem. 28(21): 4018-22.
31. Avis, P. G., Bray, R. C., et al. (1955). " Cellular constituents. The chemistry of xanthine oxidase. Part I. The preparation of a crystalline xanthine oxidase from cow's milk." J. Chem. Soc. : 1100-6
32. Wright, R. M., Vaitaitis, M. G., et al. (1993). " cDNA cloning, characterization, and tissue-specific expression of human xanthine dehydrogenase/xanthine oxidase." Proc. Natl. Acad. Sui. U.S.A. : m. 90(22): 10690-4.
33. Kalakar, H. M., Kjeldgaard, N. O., et al. (1948). " Inhibition of xanthine oxdiase and related enzymes by 6-pteridyl aldehyde." J. Biol. Chem. 174: 771-2
34. Oettl, K.,and Reibnegger, G. (1999). " Pteridines as inhibitors of xanthine oxidase: structural requirements." Biochim. Biophys. Acta. 1430(9): 387-95
35. Lowry, O. H., Bessey, O. A., et al. (1949). " Pterine oxidase." J. Biol. Chem. 180: 399-410
36. Lorente, C., and Thomas, A. H. (2006). " Photophysics and photochemistry of pterins in aqueous solution." Acc. Chem. Res. 39(6): 395-402
37. Thomas, A. H., Capparelli, A. L., et al. (2002). " Photochemical behavior of flokic acid in alkaline aqueous slolutions and evolution of its photoproducts." Helv. Chim. Acta 85(8): 2300-15
38. Hille, R., and Massey, V. (1981). "Tight binding inhibirors of xanhine oxidase." Pharmac. Ther. 14: 249-63
39. Thomas, A., Einschlag, F. G., et al. (1998). "Frist steps in the photochemistry of folate in alkaline medium." J. Photochem. Photobiol., A. 116(4): 187-90
40. Thomas, A., Suárez, G., et al. (2000). " Study of the photolysis of folic acid and 6-formylpterin in acid aqueous solutions." J. Photochem. Photobiol., A. 135(2-3): 147-54
41. Suárez, G., Cabrerizo, F. M., et al. (2000). "Study of the photolysis of 6-carboxypterin in acid and alkaline aqueous solutions." J. Photochem. Photobiol., A. 132(1-2): 53-7
42. Thomas, A. H., Suárez, G., et al. (2001). " Photochemistry of 6-Formylpterin in Alkaline Mediums." HeIv. Chim. Acta 84(12): 3849-60
43. http://www.expasy.org/
44. 林欣霈, "新型多芽基螢光感測分子之合成與光誘發黃嘌呤氧化酶作用機制之研究", 清華大學碩士論文, 2005。
45. 李欣盈, "單一原子的化學突變對黃嘌呤氧化酶催化行為之影響", 清華大學碩士論文, 2005。
46. Thijssen, H. H. (1973). " A simple method for preparing 2-amino-4-hydroxy-6-formylpteridine, a precursor of the pteridine substrate of dihydropteroate biosynthesis." Anal. Biochem. 54(2): 609-11
47. Highbarger, L. A., Gerlt, J. A., et al. (1996). "Mechanism of the reaction catalyzed by acetoacetate decarboxylase. Importance of lysine 116 in determining the pKa of active-site lysine 115." Biochemisty 35(1): 41-6
48. Vermeersch, J. J., Christmann-Franck, S., et al. (2004). "Pyridoxal 5’-phosphate inactivates DNA topoisomerase IB by modifying the lysine general acid." Nucleic Acids Res. 32(18): 5649-57
49. Tai, L. A., and Hwang, K. C. (2004). "Cooperative catalysis in the homodimer subunits of xanthine oxidase." Biochemisty. 43(16): 4869-76
50. Fukuda, L., Sakamoto, F. (1998). " Photopromoted oxidative cyclization of an o-phenylene-bridged Schiff base via a manganese(III) complex, leading to a fluorescent compound, 2-(2-hydroxyphenyl)benzimidazole " Biochemisty. 13: 1391-2

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