臺灣博碩士論文加值系統

研究目的：本篇研究是探討心血管藥物硝基化合物（nitro compounds）和脂肪酵素lipase活化性的關係。在試管內觀察硝基化合物和假單孢菌屬脂肪酵素（Pseudomonas species lipase,PSL）交互作用的特徵。
實驗結果:苦酸PA及收斂酸styphnic acid的特性是PSL的混合型mixed-type抑制劑inhibitors。從化學結構觀點，PA及styphnic acid帶氫氧基hydroxyl讓這兩種硝基化合物具親水性（hydrophilic），所以PA及styphnic acid能進入lipase的hydrophilic active site親水催化反應位置，成為這酵素的抑制劑是因為這兩個化合物的親水性特徵。
另一方面，TNT，RDX，及HNIW是PSL基本（essential）的活化劑（activators），從化學結構的觀點，TNT，RDX，HNIW比PA及styphnic acid較具疏水性（hydrophobic），因此當TNT，RDX，HNIW混合活性界面劑triton-X 100結合到PSL的co-lipase結合位置binding site，而成為基本的PSL活化劑。
有趣的是HMX及硝化甘油既不是這酵素的抑制劑也不是活化劑，因HMX及硝化甘油的疏水性hydrophobic特徵，使它們並不結合到PSL的反應位置（active site）。有甘油主鏈的硝化甘油比起其他硝基化合物具有更穩定的構形（conformations），所以和界面活性劑作用較鬆弛。
結論：本論文主要觀察，在硝基化合物中包括心血管藥物硝化甘油存在時，假單胞屬的脂肪酵素PSL催化水解對硝基苯丁酸酯（p-nitrophenyl butyrate） 在試管內的酵素動力學研究。
依動力學來說PA 及 styphnic acid 是混合型的抑制劑，但TNT,RDX 及 HNIW 是基本的活化劑，有趣的是 HMX 及 硝化甘油既不是抑制劑也不是活化劑。

Purpose :
This dissertation embarks to understand the relationships between nitro compounds and the activation of lipase. Thus, the goal of this study is then to characterize interactions between nitro compounds and Pseudomonas Species lipase (PSL) in vitro.
Results :
PA and styphnic acid are characterized as the mixed-type inhibitors of PSL. From chemical structure point of view, PA and styphnic acid carry hydroxyl groups and make both compounds hydrophilic. Therefore, PA and styphnic acid are capable of entering the hydrophilic active site of Pseudomonas Species lipase and become inhibitors of the enzyme due to the hydrophilic character of both compounds.
On the other hand, TNT, RDX, and HNIW are the essential activators of PSL. From chemical structure point of view, TNT, RDX, and HNIW are more hydrophobic than PA and styphnic acid. Therefore, TNT, RDX, and HNIW presumably mix with triton-X 100 and bind to the co-lipase binding site of PSL and become the essential activators of PSL. Moreover, the interfacial activation of PSL by the essential activators in the presence of detergents is proposed according to the lipase-colipase mechanism. Interestingly, HMX and nitroglycerin are neither an inhibitor nor an activator of the enzyme. Therefore, HMX and nitroglycerin do not bind to the active site of PSL due to the hydrophobic characters of HMX and nitroglycerin. Moreover, HMX does not bind to the co-lipase binding site of PSL probably due to a huge dimension of HMX and weak interactions between HMX and detergent triton X-100. With the glycerol backbone, nitroglycerin has more stable conformations than other nitro compounds and therefore it interacts loosely with the detergent.
Conclusions :
The goal of this work is to determine the enzyme kinetics for Pseudomonas Species lipase catalyzed hydrolysis of substrate p-nitrophenyl butyrate in the presence of nitro compounds such as nitroglycerin, 2,4,6-trinitrotoluene (TNT), picric acid, styphnic acid, hexahydro-1,3,5-trinitrotriazocine (RDX), octahydro-1,3,5,7-tetranitrotriazocine (HMX), and hexanitrohexaazaisowurtzitane (HNIW) in vitro. Kinetically, picric acid and styphnic acid are the mixed-type inhibitors but TNT, RDX, and HNIW are the essential activators of the enzyme in the presence of a detergent triton-X 100. Interestingly, HMX and nitroglycerin are neither an inhibitor nor an activator of the enzyme.

表目錄…………………………………………………………………3
圖目錄…………………………………………………………………4
中文摘要………………………………………………………………5
英文摘要………………………………………………………………7
壹、 前言……………………………………………………………10
貳、 研究目的………………………………………………………28
參、 研究方法………………………………………………………29
肆、 研究結果和討論………………………………………………32
伍、 結論……………………………………………………………35
陸、 參考文獻………………………………………………………54
附錄 …………………………………………………………………64

1.Prescott E, scharling H, Osler M, Schnohr P. "Importance of light smoking and inhalation habits on risk of myocardial infarction and all cause mortality". A 22 year follow up of 12,149 men and women in The Copenhagen City Heart Study. J Epidemiol Community Health 56：702, 2002.
2.Howard G. Wagenknecht LE, Burke GL, et al. "Cigarette smoking and progression of atherosclerosis". The Atherosclerosis Risk in Communities （ARIC） Study. JAMA 279：119, 1998.
3.Fusegawa y, Goto s, Handa S, et al. "Platelet spontaneous aggregation in platelet-rich plasma is increased in habitual smokers". Thromb Res 93：271,1999.
4.Adams MR, Jessup W, Celermajer DS. "Cigarette smoking is associated with increased human monocyte adhesion to endothelial cells". Reversibility with oral L-arginine but not vitamin C. J Am Coll Cardiol 29：491, 1997.
5.Chobanian AV, Bakris GL, Black HR, et al."The Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure". The JNC 7 report. JAMA 289：2560, 2003.
6.Vasan RS, Larson MG, Leip EP, et al. "Impact of high-normal blood pressure on the risk of cardiovascular disease". N Engl J Med 345：1291, 2001.
7.Libby P, Aikawa M, Schonbeck U. "Cholesterol and atherosclerosis". Biochim Biophys Acta 1529：299, 2000.
8.Stamler J, Davigllus ML, Garside DB, et al. "Relationship of baseline serum cholesterol levels in 3 large cohorts of younger men to long-term coronary, cardiovascular, and allcause mortality and to longevity. JAMA 284：311, 2000.
9.Howard BV, Rodriguez BL, Bennet PH, et al. "Prevention Conference VI： Diabetes and Cardiovascular disease". Writing Group I：Epidemiology. Circulation 105：e132, 2002.
10.Gu K, Cowie CC, Harris MI. "Mortality in adults with and without diabetes in a national cohort of the U.S. population". 1971-1993. Diabetes Care 21：1138, 1998.
11.Hu FB, Stampfer MJ, Haffner SM, et al. "Elevated risk of cardiovascular disease prior to clinical diagnosis of type 2 diabetes". Diabetes Care 25：1129, 2002.
12.Gillum RF, Mussolino ME, Madans JH. "Diabetes mellitus, coronary heart disease incidence, and death from all causes in African American and European American women". The NHANES I epidemiologic follow-up study. J Clin Epidemiol 53：511, 2000.
13.Garber AJ. "Attenuating CV risk factors in patients with diabetes". Clinical evidence to clinical practice". Diabetes Obes Metab 4（Suppl 1）：S5, 2002.
14.Henry P, Thomas F, Benetos A, Guize L. "Impaired fasting glucose, blood pressure and cardiovascular disease mortality". Hypertension 40：458, 2002.
15.Kuller LH, Velentgas P, Barzilay J, et al. "Diabetes mellitus：Subclinical cardiovascular disease and risk of incident cardiovascular disease and all-cause mortality". Arterioscler Thromb Vasc Biol 20：823, 2000.
16.Ford ES, Giles WH, Dietz WH. "Prevalence of the metabolic syndrome among U.S. adults". Findings from the third National Health and Nutrition Examination Survey. JAMA 287：356, 2002.
17.Lakka HM, Laaksonen DE, Lakka TA, et al. "The metabolic syndrome and total and cardiovascular disease mortality in middle-aged men". JAMA 288：2709, 2002.
18.Manson JE, Greenland P, LaCroix AZ, et al. "Walking compared with vigorous exercise for the prevention of cardiovascular events in women". N Engl J Med 347：716, 2002.
19.Manson JE, Hu FB, Rich-Edwards JW, et al. "A prospective study of walking as compared with vigorous exercise in the prevention of coronary heart disease in women". N Engl J Med 341：650, 1999.
20.Lee IM, Rexrode KM, Cook NR, et al. "Physical activity and coronary heart disease in women". Is “no pain, no gain”passe？ JAMA 285：1447, 2001.
21.Boule NG, Haddad E, Kenny GP,et al. "Effects of exercise on glycemic control and body mass in type 2 diabetes mellitus". A meta-analysis of controlled clinical trials. JAMA 286：1218, 2001.
22.Rexrode KM, Carey VJ, Hennekens CH, et al. "Abdominal adiposity and coronary heart disease in women". JAMA 280：1843, 1998.
23.Brunner EJ, Hemingway H, Walker BR, et al. "Adrenocortical, autonomic, and inflammatory causes of the metabolic syndrome". Nested case-control study. Circulation 106：2659, 2002.
24.Hjemdahl P. "Stress and the metabolic syndrome". An interesting but enigmatic, association. Circulation 106：2634, 2002.
25.Kivimaki M, Leino-Arjas P, Luukkonen R, et al. "Work stress and risk of cardiovascular mortality". Prospective cohort study of industrial employees. BMJ 325：857, 2002.
26.Iso H, Date C, Yamamoto A, et al. "Perceived mental stress and mortality from cardiovascular disease among Japanese men and women". The Japan Collaborative Cohort Study for Evaluation of Cancer Risk Sponsored by Monbusho （JACC Study）. Circulation 106：1229, 2002.
27.Khot UN, Khot MB, Bajzer CT, et al. "Prevalence of conventional risk factors in patients with coronary heart disease". JAMA 290：898, 2003.
28.Calabro P, Willerson JT, Yeh ET. "Inflammatory cytokines stimulated C-reactive protein production by human coronary artery smooth muscle cells". Circulation 108：1930, 2003.
29.Jabs WJ, Theissing E, Nitschke M, et al. "Local generation of C-reactive protein in diseased coronary artery venous bypass grafts and normal vascular tissue". Circulation 108：1428, 2003.
30.Paul A, Ko KW, Yechoor V, et al. "C-reactive protein accelerates the progression of atherosclerosis in apolipoprotein E-deficient mice". Circulation 109：647, 2004.
31.Caplice NM, Panetta C, Peterson TE, et al. "Lipoprotein（a）binds and inactivates tissue factor pathway inhibitor". A novel link between lipoproteins and thrombosis. Blood 98：2980, 2001.
32.Buechler C, Ullrich H, Ritter M, et al. "Lipoprotein（a）up-regulates the expression of the plasminogen activator inhibitor 2 in human blood monocytes". Blood 97：981, 2001.
33.Margaglione M, Cappucci G, Colaizzo D, et al. "Fibrinogen plasma levels in an apparently healthy general population". Relation to environmental and genetic determinants. Throm Haemost 80：805, 1998.
34.Vasan RS, Beiser A, D''Agostino RB, et al. "Plasma homocysteine and risk for congestive heart failure in adults without prior myocardial infarction". JAMA 289：1251, 2003.
35.Eugene B, Douglas PZ, Peter L. "Lipoprotein disorder and cardiovascular disease". Heart Disease 1：1013, 2005
36.O''Conell BJ, Genest J Jr. "High-density lipoproteins and endothelial function". Circulation 104：1978, 2001.
37.Li XA, Titlow WB, Jackson BA, et al. "High density lipoprotein binding to scavenger receptor, class B, type I activates endothelial nitric-oxide synthase in a ceramide-dependent manner". J Biol Chem 277：11058, 2002.
38.Mooser V, Mancini FP, Bopp S, et al. "Sequence polymorphisms in the apo（a）gene associated with specific levels of Lp（a）in plasma". Hum Mol Genet 4：173, 1995.
39.Danesh J, Collins R, Peto R. "Lipoprotein（a）and coronary heart disease：Meta-analysis of prospective studies". Circulation 102：1082, 2000.
40.Sorci-Thomas MG, Curtiss L, Parks JS, et al. "The hydrophobic face orientation of apolipoprotein A-I amphipathic helix domain 143-164 regulates lecithin：cholesterol acyltransferase activation". J Biol Chem 273：11776, 1998.
41.Svendsen A (2000). "Lipase protein engineering". Biochim Biophys Acta 1543 (2): 223-228.
42.Afonso C, Tulman E, Lu Z, Oma E, Kutish G, Rock D (1999). "The genome of Melanoplus sanguinipes entomopoxvirus". J Virol 73 (1): 533-52.
43.Girod A, Wobus C, Zádori Z, Ried M, Leike K, Tijssen P, Kleinschmidt J, Hallek M (2002). "The VP1 capsid protein of adeno-associated virus type 2 is carrying a phospholipase A2 domain required for virus infectivity". J Gen Virol 83 (Pt 5): 973-8.
44.Winkler FK, D''Arcy A, and W Hunziker (1990). "Structure of human pancreatic lipase". Nature 343 (6260): 771-774.
45.Diaz, B.L., and J. P. Arm. (2003). "Phospholipase A(2).". Prostaglandins Leukot Essent Fatty Acids 2-3: 87-97.
46.Goñi F, Alonso A (2002). "Sphingomyelinases: enzymology and membrane activity". FEBS Lett 531 (1): 38-46.
47.Schrag J, Cygler M. "Lipases and alpha/beta hydrolase fold". Methods Enzymol 284: 85-107.
48.Winkler FK, D''Arcy A, and W Hunziker (1990). "Structure of human pancreatic lipase". Nature 343 (6260): 771-774.
49.Egmond, M. R., and C. J. van Bemmel (1997). "Impact of Structural Information on Understanding of Lipolytic Function". Methods Enzymol 284: 119-129.
50.Withers-Martinez C, Carriere F, Verger R, Bourgeois D, and C Cambillau (1996). "A pancreatic lipase with a phospholipase A1 activity: crystal structure of a chimeric pancreatic lipase-related protein 2 from guinea pig". Structure 4 (11): 1363-74.
51.Brady, L., A. M. Brzozowski, Z. S. Derewenda, E. Dodson, G. Dodson, S. Tolley, J. P. Turkenburg, L. Christiansen, B. Huge-Jensen, L. Norskov, and et al. (1990). "A serine protease triad forms the catalytic centre of a triacylglycerol lipase.". Nature 343 (6260): 767-70.
52.Lowe ME (1992). "The catalytic site residues and interfacial binding of human pancreatic lipase". J Biol Chem 267 (24): 17069-73.
53.Spiegel S, Foster D, and R Kolesnick (1996). "Signal transduction through lipid second messengers". Curr Opin Cell Biol 8 (2): 159-67.
54.Tjoelker LW, Eberhardt C, Unger J, Trong HL, Zimmerman GA, McIntyre TM, Stafforini DM, Prescott SM, and PW Gray (1995). "Plasma platelet-activating factor acetylhydrolase is a secreted phospholipase A2 with a catalytic triad". J Biol Chem 270 (43): 25481-7.
55.Lowe ME (2002). "The triglyceride lipases of the pancreas". J Lipid Res 43 (12): 2007-16.
56.Crenon I, Foglizzo E, Kerfelec B, Verine A, Pignol D, Hermoso J, Bonicel J, Chapus C (1998). "Pancreatic lipase-related protein type I: a specialized lipase or an inactive enzyme". Protein Eng 11 (2): 135-42.
57.De Caro J, Carriere F, Barboni P, Giller T, Verger R, De Caro A (1998). "Pancreatic lipase-related protein 1 (PLRP1) is present in the pancreatic juice of several species". Biochim Biophys Acta 1387 (1-2): 331-41.
58.Guo Z, Xu X (2005). "New opportunity for enzymatic modification of fats and oils with industrial potentials.". Org Biomol Chem 3 (14): 2615-9.
59.Gupta R, Gupta N, Rathi P (2004). "Bacterial lipases: an overview of production, purification and biochemical properties". Appl Microbiol Biotechnol 64 (6): 763-81.
60.Ban K, Kaieda M, Matsumoto T, Kondo A, Fukuda H (2001). "Whole cell biocatalyst for biodiesel fuel production utilizing Rhizopus oryzae cells immobilized within biomass support particles". Biochem Eng J 8 (1): 39-43.
61.Parker JD, Parker JO. "Nitrate therapy for stable angina pectoris". N Engl J Med 38：520-531, 1998.
62.Brown BG, Bolson E, Petersen RB, et al. "The mechanisms of nitroglycerin action：Stenosis vasodilatation as a major component of the drug response". Circulation 64：1089-1097, 1981.
63.Parker JO. "Nitrates and angina pectoris". Am J Cardiol 72：3C-6C, 1993.
64.W. Boland, C. Fröbl, N. Lorenz. Synthesis 12：1049, 1991.
65.F. Theil. Chem Rev 95：2203, 1995.
66.A. Svendsen. "Lipases. Their structure biochemistry and application". P. Woolley, SB Petersen (Eds), Cambridge University Press, Cambridge. p. 1 1994.
67.H. van Tilbeurgh, M.-P. Egloff, C. Martinez, N. Rugani, R. Verger, C. Cambillau. Nature 362：814, 1993.
68.D. A. Lang, M. L. M. Mannesse, G. H. DeHaas, H. M. Verheij, B. W. Dijkstra. Eur J Biochem 254：333, 1998.
69.J. D. Schrag, Y. Li, M. Cygler, D. Lang, T. Burgdorf, H.-J. Hecht, R. Schmid, D. Schomburg, T. J. Rydel, J. D. Oliver, L. C. Strickland, C. M. Dunaway, S. B. Larson,; J. Day, A. McPherson. Structure 5：187, 1997.
70.K. K. Kim, H. K. Song, D. H. Shin, K. Y. Hwang, S. W. Suh. Structure 5：173, 1997.
71.P. Grochulski, F. Bouthillier, R. J. Kazlauskas, A. N. Serreqi, J. D. Schrag, E. Ziomek, M. Cygler. Biochemistry 33：3494, 1994.
72.M. Luic, S. Tomic, I. Lescic, E. Liubovic, D. Sepac, V. Sunjic, L. Vitale, W. Saenger, B. Kojic-Prodic. Eur J Biochem 268：3964, 2001.
73.L. Brady, A. M. Brzozowski, Z. S. Derewenda, E. Dodson, G. Dodson, S. Tolley, J. P. Turkenburg, L. Christiansen, B. Huge-Jensen, L. Norskov, L. Thim, U. Menge. Nature 343：767, 1990.
74.J. Pleiss, M. Fischer, R. D. Schmid. Chem Phys Lipids 93：67, 1998.
75.J.-F. Cavalier, G. Buono, R. Verger. Acc Chem Res 33：579, 2000.
76.A. Svendsen. Biochim Biophys Acta 1543：223, 2000.
77.A. M. Brozozowski, U. Derewenda, Z. S. Derewenda, G. G. Dodson, D. M. Lawson, J. P. Turkenburg, F. Bjorkling, B. Huge-Jensen, S. A. Patkar, L. Thim. Nature 351：491, 1991.
78.Z. S. Derewenda. Nat Struct Biol 2：347, 1995.
79.M. Pioruńska-Stolzmann, A. Piroruńska-Mikołajczak. Pharm Res 43：359, 2002.
80.I. H. Segel. Enzyme Kinetics, Wiely, New York, 1975.
81.M.-P. Egloff, F. Marguet, G. Buono, R. Verger, C. Cambillau, H. van Tilbeurgh. Biochemistry 34：2751, 1995.
82.M.-C. Lin, C.-P. Lu, Y.-R. Cheng, Y.-F. Lin, C.-S. Lin, G. Lin. Chem Phys Lipids 146：85-93, 2007.