隨著量子力學的發展，我們逐漸認知到磁場可以影響化學反應，當系統有大於一個自由基時，外在磁場會影響其耦合自旋狀態的分配，而造成自由基對重新結合的化學選擇性，而自然界中演化的候鳥，也藉由這樣的機制在地球磁場下導航。我們於磁場下觀察離胺酸5,6胺基異位酶( Lysine 5,6-Aminomutase; 5,6-LAM )的催化速率，在7600高斯時，其催化速率降低了16%，並比對Q-band EPR的CoII 4-thialysyl• RP 譜線的模擬，顯示其各向同性交換交互作用2J = 8000gauss，表明7600高斯的磁場效應，可能是由於單重態(ΣS = 0)與三重態(ΣS = 1)能階交錯所造成，因而在6500到8500 gauss外加磁場範圍間，藉由系統間交錯而轉移至單重態的耦合自旋狀態。5,6-LAM為B12(Adenosylcobalamin; AdoCbl)與B6(Pyridoxal phosphate; PLP)的相依酵素，過去藉由4-thia-L-lysine的抑制反應，而間接的證實其為自由基引導催化酵素，其催化反應由AdoCbl的鈷碳的均裂，形成不穩定的去氧腺苷自由基(5’ -deoxyadenosyl radical; Ado•)及鈷二價(Cob(II)alamin)，Ado•藉由抓取受質五號碳的上的氫，而使自由基轉移至受質五號碳的位置上，繼續一連串的自由基連鎖反應，而催化了產物的生成。而5,6-LAM的磁場效應，顯示與自然受質的催化過程中，存在自由基的中間體，而2J = 8000G能量差，更限制精細結構交互作用與自旋改寫機制的磁場效應，J-共振引起的磁場效應，非常清楚地顯示於高磁場位置，並為酵素的磁場效應，增添了一個新的佐證。

With the development of Quantum mechanics, we have different cognize in chemical reaction. When a system has more than one radical, alteration of the coupled spin-state population by an external magnetic field may lead to changes in the chemical reaction rate through spin-selective chemistry. Migratory birds in the evolution of nature, magnetic field is beneficial to migratory birds navigation through the radical-pair mechanism. We show the reaction of lysine 5,6-aminomutase (5,6-LAM) is magnetic field dependent. The enzyme catalytic efficiency decreased 16% at 7600 gauss. It compares with simulated Q-band EPR spectrum of a transient CoII 4-thialysyl• RP intermediate in 5,6-LAM, the isotropy exchange interaction (2J) = 8000 gauss. This means that magnetic effect is Singlet (ΣS = 0)-Triplet (ΣS = 1) Energy Level Crossing at 7600gauss, application of an external magnetic field in the range of 6500 to 8500 gauss triggers intersystem crossing to the singlet {cob(II)alamin-substrate} radical-pair state. Lysine 5,6-Aminomutase (5,6-LAM) is an B12(Adenosylcobalamin; AdoCbl) and B6(Pyridoxal phosphate; PLP) dependent enzyme. The radical mechanism provides by 4-thia-L-lysine inhibitors indirectly. The catalytic from homolysis Co-C bond on AdoCbl, formatting the 5'-deoxyadenosyl radical (Ado•) and Cob(II)alamin. Ado• abstracts a C5(H) from the substrate, formatting substrate radical, product will product after free radical ripple effect. Magnetic effect of 5,6-LAM, show the reaction of nature substrate proceeds via radical pair intermediates. The energy gap is 8000gauss, Hyperfine structure interaction and Spin Rephasing mechanism are severely restricted. The magnetic field effect caused by J-resonance is clearly shown in the high magnetic field position, and added a new evidence for the magnetic field effect of enzymes.

謝誌 I
摘要 II
ABSTRACT III
目錄 IV
表目錄 VII
圖目錄 VIII
第1章 緒論 1
1.1 研究背景 1
1.2 研究動機 1
1.3 研究目的 2
第2章 文獻回顧 3
2.1 蛋白質簡介 3
2.1.1 酶簡介 3
2.1.2 輔酶 4
2.2 離胺酸5,6胺基異位酶簡介 5
2.2.1 5,6-LAM之結構 6
2.2.2 5,6-LAM之輔酵素 8
2.2.3 5,6-LAM之假設反應機制 11
2.2.4 5,6-LAM 自由基中間體 13
2.2.5 5,6-LAM之自殺失活現象 16
2.3 酵素動力學 17
2.3.1 Michaelis-Menten動力學模型 18
2.3.2 Lineweaver-Burk Plot 19
2.3.3 Eadie-Hofstee Plot 19
2.3.4 抑制作用 19
2.4 酵素的磁場效應(Magnetic field effects; MFE) 20
2.4.1 系統間交錯機制(Intersystem crossing; ISC) 22
2.4.2 腺苷鈷胺(AdoCbl)磁場效應 26
2.4.3 乙醇胺氨裂解酶(EAL)的磁場效應 27
2.4.4 候鳥磁感測器 29
第3章 實驗儀器與原理 31
3.1 層析法 31
3.1.1 快速液相層析儀(FPLC) 31
3.1.2 高效液相層析儀(HPLC) 35
3.1.3 薄層層析法(TLC) 37
3.2 電泳膠片分析法 38
3.3 紫外光-可見光分光光度法 40
3.4 電子順磁共振儀 41
3.4.1 塞曼效應(Zeeman effect) 42
3.4.2 精細結構交互作用(Hyperfine Interactions) 43
3.4.3電子-電子交互作用(Electron-Electron Interaction) 44
3.4.4 朗德g因子 45
第4章 材料與方法 47
4.1 養菌 47
4.2 破菌 48
4.3 純化 49
4.4 TLC活性檢測 52
4.5 SDS-PAGE分析 53
4.6 酵素動力學參數測定 55
4.7 EPR量測 60
第5章 結果與討論 63
5.1 純化 63
5.1.1 Phenyl疏水交換層析 63
5.1.2 QFF陰離子交換層析 64
5.2 酵素動力學參數實驗 67
5.3 磁場相依的動力學參數實驗 72
5.3.1 自然受質D-lysine 73
5.3.2 同位素受質D-lysine-4,4,5,5,-d4 75
5.4 ERR光譜分析 79
5.4.1 X-band EPR 79
5.4.2 Q-band EPR 83
5.5 討論 87
第6章 結論與總結 91
附錄1 93
中間產物自由基結構證明 93
附錄2 97
Tyrosine殘基對AdoCbl Co-C斷鍵的影響 97
參考文獻 101

[1]Banerjee, R. (1999). Chemistry and biochemistry of B12: Wiley.
[2]Banerjee, R. (2003). Radical Carbon Skeleton Rearrangements:  Catalysis by Coenzyme B12-Dependent Mutases. Chemical Reviews, 103(6), 2083-2094. doi: 10.1021/cr0204395
[3]Berkovitch, F., Behshad, E., Tang, K.-H., Enns, E. A., Frey, P. A., & Drennan, C. L. (2004). A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase. Proceedings of the National Academy of Sciences of the United States of America, 101(45), 15870-15875. doi: 10.1073/pnas.0407074101
[4]Bothe, H., Darley, D. J., Albracht, S. P. J., Gerfen, G. J., Golding, B. T., & Buckel, W. (1998). Identification of the 4-Glutamyl Radical as an Intermediate in the Carbon Skeleton Rearrangement Catalyzed by Coenzyme B12-Dependent Glutamate Mutase from Clostridium cochlearium. Biochemistry, 37(12), 4105-4113. doi: 10.1021/bi971393q
[5]Brocklehurst, B. (2002). Magnetic fields and radical reactions: recent developments and their role in nature. Chemical Society Reviews, 31(5), 301-311. doi: 10.1039/B107250C
[6]Brocklehurst, B., & McLauchlan, K. A. (1996). Free radical mechanism for the effects of environmental electromagnetic fields on biological systems. Int J Radiat Biol, 69(1), 3-24.
[7]Buchachenko, A. (2016). Why magnetic and electromagnetic effects in biology are irreproducible and contradictory? Bioelectromagnetics, 37(1), 1-13. doi: 10.1002/bem.21947
[8]Canfield, J. M., & Warncke, K. (2005). Active Site Reactant Center Geometry in the CoII−Product Radical Pair State of Coenzyme B12-Dependent Ethanolamine Deaminase Determined by Using Orientation-Selection Electron Spin−Echo Envelope Modulation Spectroscopy. The Journal of Physical Chemistry B, 109(7), 3053-3064. doi: 10.1021/jp046167m
[9]Chagovetz, A. M., & Grissom, C. B. (1993). Magnetic field effects in adenosylcob(III)alamin photolysis: relevance to B12 enzymes. Journal of the American Chemical Society, 115(25), 12152-12157. doi: 10.1021/ja00078a063
[10]Chang, C. H., & Frey, P. A. (2000). Cloning, Sequencing, Heterologous Expression, Purification, and Characterization of Adenosylcobalamin-dependentd-Lysine 5,6-Aminomutase from Clostridium sticklandii. Journal of Biological Chemistry, 275(1), 106-114. doi: 10.1074/jbc.275.1.106
[11]Chen, Y.-H., Maity, A. N., Frey, P. A., & Ke, S.-C. (2013). Mechanism-based Inhibition Reveals Transitions between Two Conformational States in the Action of Lysine 5,6-Aminomutase: A Combination of Electron Paramagnetic Resonance Spectroscopy, Electron Nuclear Double Resonance Spectroscopy, and Density Functional Theory Study. Journal of the American Chemical Society, 135(2), 788-794. doi: 10.1021/ja309603a
[12]Chen, Y.-H., Maity, A. N., Pan, Y.-C., Frey, P. A., & Ke, S.-C. (2011). Radical Stabilization Is Crucial in the Mechanism of Action of Lysine 5,6-Aminomutase: Role of Tyrosine-263α As Revealed by Electron Paramagnetic Resonance Spectroscopy. Journal of the American Chemical Society, 133(43), 17152-17155. doi: 10.1021/ja207766c
[13]Christiane, R. T., & Kevin, B. H. (2004). A Study of Spin Chemistry in Weak Magnetic Fields. Philosophical Transactions: Mathematical, Physical and Engineering Sciences, 362(1825), 2573-2589.
[14]contributors, W. (8 March 2018 23:18 UTC). Agarose gel electrophoresis. Retrieved 1 May 2018 05:54 UTC, from https://en.wikipedia.org/w/index.php?title=Agarose_gel_electrophoresis&oldid=829489707
[15]contributors, W. (30 November 2017 12:21 UTC). Fast protein liquid chromatography. Retrieved 1 May 2018 05:13 UTC, from https://en.wikipedia.org/w/index.php?title=Fast_protein_liquid_chromatography&oldid=812880668
[16]contributors, W. (4 March 2018 20:19 UTC). High-performance liquid chromatography. Retrieved 1 May 2018 05:19 UTC, from https://en.wikipedia.org/w/index.php?title=High-performance_liquid_chromatography&oldid=828794783
[17]contributors, W. (26 April 2018 17:19 UTC). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Retrieved 1 May 2018 05:50 UTC, from https://en.wikipedia.org/w/index.php?title=SDS-PAGE&oldid=838384288
[18]contributors, W. (17 April 2018 04:13 UTC). Thin-layer chromatography. Retrieved 1 May 2018 05:32 UTC, from https://en.wikipedia.org/w/index.php?title=Thin-layer_chromatography&oldid=836840837
[19]contributors, W. (20 April 2018 20:50 UTC). Ultraviolet-Vvisible spectroscopy. Retrieved 1 May 2018 06:00 UTC, from https://en.wikipedia.org/w/index.php?title=Ultraviolet%E2%80%93visible_spectroscopy&oldid=837440884
[20]Eichwald, C., & Walleczek, J. (1996). Model for magnetic field effects on radical pair recombination in enzyme kinetics. Biophysical Journal, 71(2), 623-631.
[21]Frey, P. A. (2010). Comprehensive Natural Products II: Chemistry and Biology: Elsevier, Oxford.
[22]Frey, P. A., Hegeman, A. D., & Reed, G. H. (2006). Free Radical Mechanisms in Enzymology. Chemical Reviews, 106(8), 3302-3316. doi: 10.1021/cr050292s
[23]Grissom, C. B. (1995). Magnetic Field Effects in Biology: A Survey of Possible Mechanisms with Emphasis on Radical-Pair Recombination. Chemical Reviews, 95(1), 3-24. doi: 10.1021/cr00033a001
[24]Haberkorn, R., & Michel-Beyerle, M. E. (1979). On the mechanism of magnetic field effects in bacterial photosynthesis. Biophysical Journal, 26(3), 489-498.
[25]Harkins, T. T., & Grissom, C. B. (1995). The Magnetic Field Dependent Step in B12 Ethanolamine Ammonia Lyase Is Radical-Pair Recombination. Journal of the American Chemical Society, 117(1), 566-567. doi: 10.1021/ja00106a079
[26]Harkins, T. T., & Grissom, C. B. (1994). Magnetic field effects on B12 ethanolamine ammonia lyase: evidence for a radical mechanism. Science, 263(5149), 958. doi: 10.1126/science.8310292
[27]Healthcare, G. (2012). ÄKTA™ pure User Manual (1 ed.): General Electric Company.
[28]Healthcare, G. (2006). Hydrophobic Interaction and Reversed Phase Chromatography Principles and Methods: General Electric Company.
[29]Healthcare, G. (2004). Ion Exchange Chromatography Principles and Methods: General Electric Company.
[30]Heinrikson, R. L., & Meredith, S. C. (1984). Amino acid analysis by reverse-phase high-performance liquid chromatography: Precolumn derivatization with phenylisothiocyanate. Analytical Biochemistry, 136(1), 65-74. doi: 10.1016/0003-2697(84)90307-5
[31]Hore, P. J., & Mouritsen, H. (2016). The Radical-Pair Mechanism of Magnetoreception. Annual Review of Biophysics, 45(1), 299-344. doi: 10.1146/annurev-biophys-032116-094545
[32]Jia, S., Liu, Y., Wu, S., & Wang, Z. (2009). Effect of static magnetic field on α-amylase activity and enzymatic reaction. Transactions of Tianjin University, 15(4), 272-275. doi: 10.1007/s12209-009-0048-8
[33]Jones, A. (2016). Magnetic field effects in proteins (Vol. 114).
[34]Jones Alex, R., Hardman Samantha, J. O., Hay, S., & Scrutton Nigel, S. (2011). Is There a Dynamic Protein Contribution to the Substrate Trigger in Coenzyme B12‐Dependent Ethanolamine Ammonia Lyase? Angewandte Chemie International Edition, 50(46), 10843-10846. doi: 10.1002/anie.201105132
[35]Jones, A. R., Hay, S., Woodward, J. R., & Scrutton, N. S. (2007). Magnetic Field Effect Studies Indicate Reduced Geminate Recombination of the Radical Pair in Substrate-Bound Adenosylcobalamin-Dependent Ethanolamine Ammonia Lyase. Journal of the American Chemical Society, 129(50), 15718-15727. doi: 10.1021/ja077124x
[36]Jones, A. R., Woodward, J. R., & Scrutton, N. S. (2009). Continuous Wave Photolysis Magnetic Field Effect Investigations with Free and Protein-Bound Alkylcobalamins. Journal of the American Chemical Society, 131(47), 17246-17253. doi: 10.1021/ja9059238
[37]Ke, S. C. (2003). Spin–spin interaction in ethanolamine deaminase. Biochimica et Biophysica Acta (BBA) - General Subjects, 1620(1), 267-272. doi: 10.1016/S0304-4165(03)00006-0
[38]Kumar, M., & Kozlowski, P. M. (2009). Role of Tyrosine Residue in the Activation of Co−C Bond in Coenzyme B12-Dependent Enzymes: Another Case of Proton-Coupled Electron Transfer? The Journal of Physical Chemistry B, 113(27), 9050-9054. doi: 10.1021/jp903878y
[39]Lo, H.-H., Lin, H.-H., Maity, A. N., & Ke, S.-C. (2016). The molecular mechanism of the open-closed protein conformational cycle transitions and coupled substrate binding, activation and product release events in lysine 5,6-aminomutase. Chemical Communications, 52(38), 6399-6402. doi: 10.1039/C6CC01888B
[40]Maity, A. N., Hsieh, C.-P., Huang, M.-H., Chen, Y.-H., Tang, K.-H., Behshad, E., . . . Ke, S.-C. (2009). Evidence for Conformational Movement and Radical Mechanism in the Reaction of 4-Thia-l-lysine with Lysine 5,6-Aminomutase. The Journal of Physical Chemistry B, 113(36), 12161-12163. doi: 10.1021/jp905357a
[41]Maity, A. N., & Ke, S.-C. (2015). 4′-CyanoPLP presents better prospect for the experimental detection of elusive cyclic intermediate radical in the reaction of lysine 5,6-aminomutase. Biochemical and Biophysical Research Communications, 457(2), 161-164. doi: 10.1016/j.bbrc.2014.12.076
[42]Maity, A. N., & Ke, S.-C. (2013). 5-Fluorolysine as alternative substrate of lysine 5,6-aminomutase: A computational study. Computational and Theoretical Chemistry, 1022, 1-5. doi: 10.1016/j.comptc.2013.08.007
[43]Maity, A. N., Lin, H.-H., Chiang, H.-S., Lo, H.-H., & Ke, S.-C. (2015). Reaction of Pyridoxal-5′-phosphate-N-oxide with Lysine 5,6-Aminomutase: Enzyme Flexibility toward Cofactor Analog. ACS Catalysis, 5(5), 3093-3099. doi: 10.1021/acscatal.5b00671
[44]Makins, C., Whitelaw, D. A., Mu, C., Walsby, C. J., & Wolthers, K. R. (2014). Isotope Effects for Deuterium Transfer and Mutagenesis of Tyr187 Provide Insight into Controlled Radical Chemistry in Adenosylcobalamin-Dependent Ornithine 4,5-Aminomutase. Biochemistry, 53(33), 5432-5443. doi: 10.1021/bi5006706
[45] Mansoorabadi, S. O., Padmakumar, R., Fazliddinova, N., Vlasie, M., Banerjee, R., & Reed, G. H. (2005). Characterization of a Succinyl-CoA Radical−Cob(II)alamin Spin Triplet Intermediate in the Reaction Catalyzed by Adenosylcobalamin-Dependent Methylmalonyl-CoA Mutase. Biochemistry, 44(9), 3153-3158. doi: 10.1021/bi0482102
[46]Manzerova, J., Krymov, V., & Gerfen, G. J. (2011). Investigating the intermediates in the reaction of ribonucleoside triphosphate reductase from Lactobacillus leichmannii: An application of HF EPR–RFQ technology. Journal of Magnetic Resonance, 213(1), 32-45. doi: 10.1016/j.jmr.2011.08.030
[47]Pierik, A. J., Ciceri, D., Lopez, R. F., Kroll, F., Bröker, G., Beatrix, B., . . . Golding, B. T. (2005). Searching for Intermediates in the Carbon Skeleton Rearrangement of 2-Methyleneglutarate to (R)-3-Methylitaconate Catalyzed by Coenzyme B12-Dependent 2-Methyleneglutarate Mutase from Eubacterium barkeri. Biochemistry, 44(31), 10541-10551. doi: 10.1021/bi050049n
[48]Reed, G. H., & Mansoorabadi, S. O. (2003). The positions of radical intermediates in the active sites of adenosylcobalamin-dependent enzymes. Current opinion in structural biology, 13(6), 716-721.
[49]Schulten, K., & Bittl, R. (1986). Probing the dynamics of a polymer with paramagnetic end groups by magnetic fields. The Journal of Chemical Physics, 84(9), 5155-5161. doi: 10.1063/1.450668
[50]Solov'yov, I. A., & Schulten, K. (2009). Magnetoreception through Cryptochrome May Involve Superoxide. Biophysical Journal, 96(12), 4804-4813. doi: 10.1016/j.bpj.2009.03.048
[51]Stadtman, T. C., & White, F. H. (1954). Tracer studies on ornithine, lysine, and formate metabolism in an amino acid fermenting Clostridium. J Bacteriol, 67(6), 651-657.
[52]Stadtman Thressa, C. Lysine Metabolism by Clostridia.
[53]Steiner, U. E., & Ulrich, T. (1989). Magnetic field effects in chemical kinetics and related phenomena. Chemical Reviews, 89(1), 51-147. doi: 10.1021/cr00091a003
[54]Stoll, S., & Schweiger, A. (2006). EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. Journal of Magnetic Resonance, 178(1), 42-55. doi: 10.1016/j.jmr.2005.08.013
[55]Stoneham, A M., Gauger, Erik M., Porfyrakis, K., Benjamin, Simon C., & Lovett, Brendon W. (2012). A New Type of Radical-Pair-Based Model for Magnetoreception. Biophysical Journal, 102(5), 961-968. doi: 10.1016/j.bpj.2012.01.007
[56]Tang, K.-H., Casarez, A. D., Wu, W., & Frey, P. A. (2003). Kinetic and biochemical analysis of the mechanism of action of lysine 5,6-aminomutase. Archives of Biochemistry and Biophysics, 418(1), 49-54. doi: 10.1016/S0003-9861(03)00346-1
[57]Tang, K.-H., Chang, C. H., & Frey, P. A. (2001). Electron Transfer in the Substrate-Dependent Suicide Inactivation of Lysine 5,6-Aminomutase. Biochemistry, 40(17), 5190-5199. doi: 10.1021/bi010157j
[58]Tang, K.-H., Mansoorabadi, S. O., Reed, G. H., & Frey, P. A. (2009). Radical Triplets and Suicide Inhibition in Reactions of 4-Thia-d- and 4-Thia-l-lysine with Lysine 5,6-Aminomutase. Biochemistry, 48(34), 8151-8160. doi: 10.1021/bi900828f
[59]Taoka, S., Padmakumar, R., Grissom Charles, B., & Banerjee, R. (1998). Magnetic field effects on coenzyme B12‐dependent enzymes: Validation of ethanolamine ammonia lyase results and extension to human methylmalonyl CoA mutase. Bioelectromagnetics, 18(7), 506-513.
[60]Tseng, C.-H., Yang, C.-H., Lin, H.-J., Wu, C., & Chen, H.-P. (2007). The S subunit of d-ornithine aminomutase from Clostridium sticklandii is responsible for the allosteric regulation in d-α-lysine aminomutase. FEMS Microbiology Letters, 274(1), 148-153. doi: 10.1111/j.1574-6968.2007.00820.x
[61]Weber, R. T., Jiang, J., & Barr, D. P. (1998). EMX USER’S MANUAL (2.0 ed. Vol. Manual Version 2.0): Bruker Instruments, Inc.
[62]Wiltschko, R., Stapput, K., Bischof, H. J., & Wiltschko, W. (2007). Light-dependent magnetoreception in birds: increasing intensity of monochromatic light changes the nature of the response. Front Zool, 4, 5. doi: 10.1186/1742-9994-4-5
[63]Wiltschko, W., Freire, R., Munro, U., Ritz, T., Rogers, L., Thalau, P., & Wiltschko, R. (2007). The magnetic compass of domestic chickens, Gallus gallus. Journal of Experimental Biology, 210(13), 2300. doi: 10.1242/jeb.004853
[64]Woodward, J. R. (2002). RADICAL PAIRS IN SOLUTION. Progress in Reaction Kinetics and Mechanism, 27(3), 165-207. doi: 10.3184/007967402103165388
[65]Woodward, Jonathan R., Foster, Timothy J., Jones, Alex R., Salaoru, Adrian T., & Scrutton, Nigel S. (2009). Time-resolved studies of radical pairs. Biochemical Society Transactions, 37(2), 358. doi: 10.1042/BST0370358