果蠅多巴胺乙醯基轉移酶的基質通道大小對酵素活性之影響

簡易檢索 / 詳目顯示

回結果列表

研究生：	楊淳窚 Yang, Chun Cheng
論文名稱：	果蠅多巴胺乙醯基轉移酶的基質通道大小對酵素活性之影響 The size effect of substrate-entrance tunnel of Dopamine N-acetyltransferase on its enzyme activity
指導教授：	呂平江 Lyu, Ping Chiang 彭明德 Perng, Ming Der
口試委員:	蘇士哲鄭惠春
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 分子醫學研究所 Institute of Molecular Medicine
論文出版年：	2015
畢業學年度：	103
語文別：	中文
論文頁數：	71
中文關鍵詞：	果蠅蛋白、苯烷基胺乙醯基轉移酶、多巴胺乙醯基轉移酶、乙醯輔酶A 、受質專一性、酵素動力學、通道瓶頸
外文關鍵詞：	Drosophila Protein, Arylalkylamine N-acetyltransferase, Dopamine N-acetyltransferase, Acetyl Coenzyme A, Substrate Specificity, Enzyme Kinetics, Tunnel bottleneck
相關次數：	點閱：2 下載：0
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

果蠅的多巴胺乙醯基轉移酶 (Dopamine N-Acetyltransferase, Dat)，是屬於苯烷基胺乙醯基轉移酶家族中的蛋白酶(EC 2.3.1.87, Arylalkylamine N-Acetyltransferase, AANAT)，它們可以催化乙醯輔酶A (Acetyl-CoA) 的乙醯基團轉移至苯烷基胺上，而產出N-乙醯苯烷基胺。近期以來，我們實驗室已經解出多巴胺乙醯基轉移酶晶體、多巴胺乙醯基轉移酶/乙醯輔酶A複合晶體兩者之蛋白質結構。隨後更進一步解出解析度高達1.2 Å的多巴胺乙醯基轉移酶的三元複合晶體 (乙醯基轉移酶/乙醯基輔酶A/基質) 的蛋白質結構。藉由此三元複合體的結構加上先前實驗室所做的等溫滴定微量熱法實驗，我們發現乙醯基轉移酶需要先結合乙烯基輔酶A，才能夠與基質作用。因此我們發現基質與乙醯基輔酶A進入乙醯基轉移酶的入口是不相同的。在先前的研究，我們實驗室發現M121以及D142坐落於基質通道的最狹窄處 (通道瓶頸)。當M121以及D142分別突變成色胺酸，經由酵素動力學實驗發現M121W以及D142W會阻礙基質進入到疏水性的基質結合空腔，進而證實通道的存在。在本實驗中，我們將M121以及D142分別突變成丙氨酸，使得通道瓶頸的直徑變大。另外也將M121以及D142一併突變成色胺酸，使得通道瓶頸變得比個別突變還小。經由酵素動力學的實驗，我們發現讓通道瓶頸變寬的D142A，與wt-Dat有著幾乎相同的活性，且對不同大小的測試基質的選擇性不如wt-Dat顯著。而讓通道瓶頸變更窄的M121WD142W的酵素活性則是有顯著降低的現象，從這結果我們可以發現通道瓶頸的大小確實會影響酵素的活性。然而，M121A雖然使得通道瓶頸變大，卻造成酵素活性顯著降低。使用LIGPLOT和PyMol進行結構分析後，我們發現M121對基質可以產生S/π作用，所以當M121被改變成丙氨酸時，改變得不單純只是通道瓶頸的大小，而是直接影響基質結合。

Drosophila dopamine N-acetyltransferase (Dat, EC 2.3.1.87) is an arylalkylamine N-acetyltransferase (AANAT), which can catalyze acetyl transfer from acetyl-coenzyme A to arylalkylamine, yielding N-acetylarylalkylamine. According to our X-ray structure, a tunnel was inside the protein with entrances at top and bottom. It has also been shown that the substrate located at the middle of Dat, and CoA located at the bottom which seemed to completely block the entrance. Dat exhibits an ordered sequential mechanism, with acetyl-CoA binding first, followed by substrate. Therefore, substrate should access to its binding site through the other entrance, and it looked like a tunnel existed. M121 and D142 located in the narrowest site of the tunnel (tunnel bottleneck). The replacement of these two residues with tryptophan resulted in a decrease in enzyme activity which may imply the hindrance to the substrate entrance by M121W and D142W. In the present study, the size effect of the substrate tunnel was on enzyme activity. We replaced M121 and D142 to alanine individually, which cause the tunnel bottleneck broader. We also made a double mutation, M121WD142W, to make the tunnel bottleneck even narrower than single mutation. The enzyme kinetic studies demonstrated that D142A had almost the same enzyme activity as that of wild type, and M121WD142W showed a significant decrease in the enzyme activity compared to wild type, M121W and D142W. These results confirmed that the size of tunnel bottleneck may affect the substrate specificity again. Although the tunnel bottleneck is broader, M121A exhibits a decrease in enzyme activity. After analysis by LIGPLOT and PyMol, we found that M121 should participate in substrate binding via S/π interactions. Therefore, changing M121 to alanine not only change the size of substrate tunnel bottleneck, but also was expected to significantly decrease the substrate binding.

中文摘要    2
Abstract    3
Content    4
Abbreviations    7
Keywords    8
Chapter 1. Introduction    9
1.1    Dopamine N-acetyltransferase (Dat) as an AANAT from Drosophila melanogaster    9
1.2    The overall structure of Drosophila Dopamine N-acetyltransferase    10
1.3    Substrate-entry tunnel of Dopamine N-Acetyltransferase    11
1.4    Motivation of the study    12
Chapter 2. Materials and Methods    13
2.1 Measurement and determination of the tunnel size of Dat    13
2.2 Protein Expression and Purification    13
2.2.1 Protein construction, Recombinant and Mutagenesis of Dat    13
2.2.2 Protein Expression of wild type Dat and mutants    14
2.2.3 Protein purification of wild type Dat and mutants    15
2.3 SDS-PAGE    15
2.4 Quantification of protein concentration    16
2.5 Identification of protein purity and molecular weight    16
2.6 Circular Dichroism Spectroscopy    17
2.7 Isothermal Titration Calorimetry binding assay    18
2.8 DTNB-based function assay    18
Chapter 3. Results and Discussion    20
3.1    Designing the mutants and measuring the changes in simulated mutants structures    20
3.2    Expression and Purification of wild-type Dat (wt-Dat) and its mutants    20
3.3    Secondary structures of wt-Dat and its mutants    21
3.4    Isothermal Titration Calorimetry Binding Assay    21
3.5    DTNB-based functional assay    23
Chapter 4. Conclusion    25
List of Tables    26
Table 2.1 Primer sequences and conditions for PCR amplification of Drosophila Dat    26
Table 3.1 ITC data for titration of wild type Dat and its mutants with dopamine    27
Table 3.2 Kinetic parameters of wild type Dat    28
Table 3.3 Kinetic parameters of D142A    29
Table 3.4 Kinetic parameters of M121WD142W    30
Table 3.5 Kinetic parameters of M121A    31
Table 3.6 Comparison of the relative enzyme activity of wild type and mutants    32
Table 3.7 Comparison of ITC data with M121W and D142W    33
List of Figures    34
Figure 1.1 The enzymatic reaction and biochemical pathway for the biosynthesis of melatonin.    34
Figure 1.2 The pleiotropic effect of melatonin    35
Figure 1.3 Emotional competence rhythms regulate by neurotransmitters in brain    36
Figure 1.4 Topology of GNATs superfamily and the binary complex structure of Dat (Dat/Acetyl-CoA)    37
Figure 1.5 The catalytic mechanism of Dat.    38
Figure 1.6 The binary complex structure of Dat (Dat/Acetyl-CoA)    39
Figure 1.7 The overall structure of the ternary complex of Dat    40
Figure 1.8 Acetyl-CoA and substrate entrances of Dat tunnel    41
Figure 1.9 The tunnel size of wild type Dat calculated by Caver software    42
Figure 1.10 Sequence alignment of insect AANATs    43
Figure 2.1 Map of the plasmid pGEX-6p3 used for the characterization of Dat    44
Figure 2.2 The catalytic reaction of AANAT based on DTNB functional assay    45
Figure 3.1 The sizes of tunnel bottleneck in M121A and D142A calculated by Caver software    46
Figure 3.2 The sizes of tunnel bottleneck in M121WD142W calculated by Caver software    47
Figure 3.3 Purification results of wild type Dat via ÄTKA prime system    48
Figure 3.4 Expression and purification of wt-Dat monitored by 13% SDS-PAGE    49
Figure 3.5 Expression and purification of mutants monitored by 13% SDS-PAGE    50
Figure 3.6 Mass spectra of mutants    51
Figure 3.7 The circular dichroism spectra of Dat and mutants    52
Figure 3.8 ITC profiles of (A) wild type Dat, (B) M121A, (C) D142A and (D) M121WD142W binding to dopamine.    53
Figure 3.9 S/π interactions between M121 and substrates of Dat    54
Figure 3.10 The potential association between Tyr64, Lys144 and Asp142    55
Figure 3.11 Catalytic efficiency of wild type Dat and its mutants    56
Figure 3.12 DTNB-based functional assay: wt-Dat, M121A, D142A and M121WD142W    57
Figure 3.13 The influence of the substrate entrance tunnel of D142A and D142W    58
Appendix I: DTNB-based functional assay of M121W and D142W    59
Appendix II: ITC data for titration of WT, M121W and D142W with dopamine.    60
Appendix III: DTNB-based functional assay of Y64A    61
Appendix IX: ITC data for titration of Y64 with dopamine,    62


                                

Ashley L. Ringer, Anaseasia Senenko, and C. David Sherrill (2007). ” Models of S/π interactions in protein structures: Comparison of the H2S–benzene complex with PDB data.” Protein Science 16:2216–2223.
Beaudette, N. V., and Langerman, N. (1978). “An improved method for obtaining thermal titration curves using micromolar quantities of protein.” Anal. Biochem. 90, 693–704.
Blenau, W., and Baumann, A. (2001). “Molecular and pharmacological properties of insect biogenic amine receptors: Lessons from Drosophila melanogaster and Apis mellifera.” Archives of Insect Biochemistry and Physiology 48, 13-38.
Bina Z-P, Sharron B-Z, et al. (2011). “Molecular evolution of multiple Arylalkylamine N-acetyltransferase (AANAT) in fish.” Mar. Drugs 9: 906-921.
Christensen, J. J., Izatt, R. M., et al. (1966). “Entropy titration. A calorimetric method for the determination of ΔG, ΔH and ΔS from a single thermometric titration.” J. Phys. Chem. 70, 2003-2010.
Christensen, J. J., Izatt, R. M., and Eatough, D. (1965). “Thermodynamics of metal cyanide coordination. V. Log K, ΔHo, and ΔSo values for the Hg2+ -cn-system.” Inorg. Chem. 4, 1278–1280.
Christensen, J. J., Wrathall, D. P., Oscarson, J. L., and Izatt, R. M. (1968). “Theoretical evaluation of entropy titration method for calorimetric determination of equilibrium constants in aqueous solution.” Anal. Chem. 40, 1713–1717.
Coto-Montes, A. and R. Hardeland (1999). "Antioxidative effects of melatonin in Drosophila melanogaster: antagonization of damage induced by the inhibition of catalase." J Pineal Res 27(3): 154-158.
Cockroft, SL; Hunter, CA; Lawson, KR; Perkins, J; Urch, CJ (2005). "Electrostatic control of aromatic stacking interactions". J. Am. Chem. Soc. 127 (24): 8594–8595.
David C. Klein, S. L. C., Patrick H. Roseboom, J.L. Weller (1997) “The Melatonin Rhyme-generating Enzyme: Molecular Regulation of Serotonin N-acetyltransferase in the Pineal Gland. Hormone research 52, 307-358.
Daniel R. Dempsey, K. A. Jeffriex et al. (2014) “Mechanistic and structural analysi of Drosophila melanogaster arylalkylamine N-acetyl transferase.” Biochemistry, 53, 7777-7793.
Dyda, F., D. C. Klein, et al. (2000). "GCN5-related N-acetyltransferases: a structural overview." Annu Rev Biophys Biomol Struct 29: 81-103.
Dr. Richard J. Wurtman, J. J. W. (1995). “Brain serotonin, carbohydrate-craving, obesity and depression.” Obesity 3, 477S-480S.
Eatough, D. (1970). “Calorimetric determination of equilibrium constants for very stable metal-ligand complexes.” Anal. Chem. 42, 635–639.
Ekmekcioglu, C. (2006). "Melatonin receptors in humans: biological role and clinical relevance." Biomed Pharmacother 60(3): 97-108.
Emmanuel A. Meyer, Ronald K. Castellano, and Franois Diederich (2003). “Interactions with Aromatic Rings in Chemical and Biological Recognition.” Angew. Chem. Int. Ed. 2003, 42, No. 11.
E.K. Perry, R. H. P., J.M. Candy et al. (1984). “Cortical serotonin-S2 receptor binding abnormalities in patients with Alzheimer’s disease: Comparisons with Parkinson’s disease.” Neuroscience 51, 353-357.
Ferry, G., C. Ubeaud, et al. (2004). "Purification of the recombinant human serotonin N-acetyltransferase (EC 2.3.1.87): further characterization of and comparison with AANAT from other species." Protein Expr Purif 38(1): 84-98.
G. A. Smythe, L. L. (1973). “Growth Hormone Regulation by Melatonin and Serotonin.” Nature 244, 355-359.
Golden RN, G. J., Corrigan MH et al. (1991) “Serotonin, suicide and aggression: clinical studies.” J Clin Psychiatry 52, 61-70.
Gogstad, G. O. and Krutnes, M. B. (1982). “ Mesurement of protein in cell suspensions using the Commassie brilliant blue dye-binding assay.” Analytical Biochemistry 126, 355-359.
Green, A. R. (1985). “Neuropharmacology of serotonin: Oxford University Press Oxford.”

Hickman AB, Klein DC, Dyda F. (1999). “Melatonin biosyntheses: the structure of serotonin N-acetyltransferase at 2.5 ˚A resolution suggests a catalytic mechanism.” Mol. Cell. 3:23–32.
Hickman, A. B., M. A. Namboodiri, et al. (1999). "The structural basis of ordered substrate binding by serotonin N-acetyltransferase: enzyme complex at 1.8 Å resolution with a bisubstrate analog." Cell 97(3): 361-369.
Hardeland, R., S. R. Pandi-Perumal, et al. (2006). "Melatonin." Int J Biochem Cell Biol 38(3): 313-316.
Hardeland, R., & Poeggeler, B. (2003). “Non-vertebrate melatonin.” J.Pineal Res 34, 233–241.
Hardeland R, Balzer I, Poeggeler B et al. (1995). “On the primary functions of melatonin in evolution: mediation of photoperiodic signals in a unicell, photooxidation and scavenging of free radicals.” J Pineal Res 18:104–111.
Hintermann, E., N. C. Grieder, et al. (1996). "Cloning of an arylalkylamine N-acetyltransferase (aaNAT1) from Drosophila melanogaster expressed in the nervous system and the gut." Proc Natl Acad Sci U S A 93(22): 12315-12320.
Hunter, Christopher A.; Sanders, Jeremy K. M. (1990). "The nature of .pi.-.pi. Interactions". J. Am. Chem. Soc. 112 (14): 5525–5534.
Hugues Ouellet, Larissa M.P., Paul R. O. de M. (2008). "Mycobacterium tuberculosis CYP130 crystal structure, biophysical characterization, and interactions with antifungal azole drugs." Journal of Biological Chemistry 283(8): 5069-5080.
I. Mayer, C. Bornestaf, et al. (1997) “Melatonin in non-mammalian vertebrates Physiological role in reproduction?” Biochem. Physiol. 188A(3): 515-531.
Jr. , W. C. J. (1990). “Protein secondary structure and circular dichroism: A practical guide.” Proteins, 205-214.
Jacqueline De Angelis, J. G., David C. Klein, and Philip A. Cole (1998). "Kinetic Analysis of catalytic mechanism of serotonin N-acetyltransferase (EC 2.3.1.87)." Journal of Biological Chemistry 273(5): 3045-3050.
Kuo-Chang Cheng, Jhen-Ni Liao and Ping-Chiang Lyu. Biochem. J, (2012), 446, 395-404.
Eva Chovancova, Antonin Pavelka, Petr Benes et al. (2012). “CAVER 3.0: A Tool for the Analysis of Transport Pathways in Dynamic Protein Structures.” PLOS Computational Biology, volume 8, issue 10, e1002708.
Kozlikova B., Sebestova E., Sustr V. (2014) “CAVER Analyst 1.0: graphic tool for interactive visualization and analysis of tunnels and channels in protein structures.”
Khalil, E. M., J. De Angelis, et al. (1998). "Indoleamine analogs as probes of the substrate selectivity and catalytic mechanism of serotonin N-acetyltransferase." J Biol Chem 273(46): 30321-30327.
L. Finocchiaro, J. C., J. M. Launay et al. (1998). “Melatonin Biosynthesis in Drosophila: Its Nature and Its Effects.” J Neurochemistry 50, 382-387.
Lerner, A. B., Case, J. D., & Takahashi, Y. (1958). “Isolation of melatonin, a pineal factor that lightens melanocytes.” J. Am. Chem. Soc 80, 2057–2058.
Macchi MM, Bruce JN. (2004). “Human pineal physiology and functional significance of melatonin.” Front Neuroendocrinol 25:177–95.
McGaughey, GB; Gagné, M; Rappé, AK (1998). "Pi-Stacking interactions. Alive and well in proteins". J. Biol. Chem. 273 (25): 15458–15463.
McMillan, W. O., Mpnteiro et al. (2002). “Development and evolution on the wing” Trends in Ecology & Evolution 17, 125-133.
Osanai-Futahashi, M., Ohde et al. (2012). “A visible dominant marker for insect transgenesis.” Nat Commun3, 1295.
Pevet, B. V.-R. a. P. (1993). "Melatonin: prescence and formation in invertebrates." Experientia 49.
Peter Karlson, C. E. S. (1962). “N-acetyl-dopamine as sclerization agent of insect cuticle.” Physiolog Chem 195, 183-184.
Pierre Blier, C.D. M. (1999). “Serotonin and Drug-Induced therapeutic Responses in Major Depression, Obsessive-Compulsive and Panic Disorders.” Neuropsychopharmacology 21, 91S-98S.
R. Hardeland. (2008). “Melatonin, hormone of darkness and more-occurrence, control mechanisms, actions and bioactive metabolites.” Cell. Mol. Life Sci 65, 2001 – 2018
Reiter RJ. (1993). “The melatonin rhythm: both a clock and a calendar.” Experientia 49: 654-664
Raffa, S. D. S. a. R. B. (2004). "Isothermal Titration Calorimetric study of RNase-A kinetics (cCMP3’-CMP) involving end-product Inhibition." Pharmaceutical Research . 21: No. 9.
Savaskan E, Olivieri G, Meier F, Brydon L, Jockers R, Ravid R, et al. (2002). “Increased melatonin 1a-receptor immunoreactivity in the hippocampus of Alzheimer’s disease patients.” J Pineal Res 32:59–62.
Scheibner, K. A., J. De Angelis, et al. (2002). "Investigation of the roles of catalytic residues in serotonin N-acetyltransferase." J Biol Chem 277(20): 18118-18126.
Spink, C., and Wadso, I. (1976). “Calorimetry as an analytical tool in biochemistry and biology.” Methods Biochem. Anal. 23, 1–159.
Sinnokrot, MO; Valeev, EF; Sherrill, CD (2002). "Estimates of the ab initio limit for pi-pi interactions: The benzene dimer". J. Am. Chem. Soc. 124 (36): 10887–10893.
Simith, P. K., Krohn et al. (1985). :Mesurement of protein using bicinchoninic acid.” Analytical Biochemistry 150, 76-85.
T. S. Wiseman, S. Williston, J. F. Brandts, and L.-N. Lin. (1989) “Rapid measurement of binding constants and heats of binding using a new titration calorimeter.” Analytic. Biochem .179:131–137.
Vetting, M. W., S. d. C. LP, et al. (2005). "Structure and functions of the GNAT superfamily of acetyltransferases." Arch Biochem Biophys 433(1): 212-226.
Wolf, E., J. De Angelis, et al. (2002). "X-ray crystallographic studies of serotonin N-acetyltransferase catalysis and inhibition." J Mol Biol 317(2): 215-224.
Woody, R. W. (1995). “Circilar dichroism. In Methods in Enzymology, S. Kenneth, ed.” Academic Press, 34-71.
Wright TR. (1987). “The genetics of biogenic amine metabolism, sclerotization, and melanization in Drosophila melanogaster.” Adv Genet 24: 127-222
Watt, G. D. (1990). “A microcalorimetric procedure for evaluating the kinetic parameters of enzymecatalyzed reactions: Kinetic measurements of the nitrogenase system.” Anal. Biochem. 187, 141–146.
Waddell, S., Armstrong et al. (2000) “The amnesiac Gene Product Is Expressed in Two Neurons in the Drosophila Brain that Are Critical for Memory.” Cell 103, 805-813.
Williams, B. A., and Toone, E. J. (1993). “Calorimetric evaluation enzyme kinetics parameters.” J. Org. Chem. 58, 3507–3510.
Wiseman, T., Williston, S., Brandts, J. F., and Lin, L. N. (1989). “Rapid measurement of binding constants and heats of binding using a new titration calorimeter.” Anal. Biochem. 179, 131–137.
Wurtman, R. J., Wurtman, J. J., Regan, M. M. et al. (2003). “Effects of normal meals rich in carbohydrates or proteins on plasma tryptophan and tyrosine ratios.” The American journal of clinical nutrtion 77, 128-132.
Yi Liang, Fen Du, et al. (2003). "Unfolding of rabbit muscle creatine kinase induced by acid." Journal of Biological Chemistry 278(32): 30098-30105.
Y.K., Byoung S.K., Y.J. Park, et al. (2010). "Crystal structure of SmcR, a Quorum-sensing master regulator of Vibrio vulnificus, provides insight into its regulation of transcription." Journal of Biological Chemistry 285(18): 14020-14030.
Young-Cho Kim, H. G. L. and Kyung-An Han (2007). “D1 Dopamine Receptor dDA1 Is Required in the Mushroom Body Neurons for Aversive and Appetitive Learning in Drosophila.” The Journal of Neuroscience 27, 7640-7647.

全文公開日期本全文未授權公開 (校內網路)
全文公開日期本全文未授權公開 (校外網路)

簡易檢索 / 詳目顯示

相關論文