在離胺酸5,6胺基異位脢(Lysine 5,6-Aminomutase；5,6-LAM)的假設反應機制中，並未能藉由自然受質D-lysine，直接證實其反應機制是透過自由基來完成催化反應，因其生命週期極為短暫。透過理論計算得知，PLP的親電性會影響中間產物自由基(I•)的穩定度，其中部分質子化的PLP， I•的能量為17.6 KJ/mol，而未質子化的PLP， I•的能量則為21.6 KJ/mol，I•的提升能使D-lysine的受質自由基累積，並有機會透過EPR觀測。
5,6-LAM中，PLP吡啶的1號氮(pyridine-N1)會與Ser238α以氫鍵交互作用連結，因serine為弱酸，會向pyridine-N1提供質子，使PLP部分質子化，因此欲藉由1-deaza-PLP及突變種S238A與5,6-LAM進行實驗上的比對，觀察PLP親電性對於5,6-LAM影響為何，並驗證理論計算的結果，然而1-deaza-PLP合成尚未完成，因此僅利用S238A與5,6-LAM進行實驗。
根據實驗結果顯示，突變種S238A與5,6-LAM的自由基耦合及精細結構交互作用，位置相互吻合，證實結構並無改變，而S238A仍無法順利捕捉D-lysine的受質自由基訊號，HPLC數據顯示S238A催化速率下降4.7倍，Transaldimination的速率為WT較快，推斷受質較易與質子化程度較高的PLP鍵結，Homolysis及Ado•於受質5號碳抓氫等過程，也受突變種S238A影響。

Since D-lysine’s lifetime is really short, we couldn’t understand the mechanism of lysine 5, 6-aminomutase (5, 6-LAM) by observing the D-lysine directly. However, the DFT shows that the electrophilicity of PLP will affect the stability of intermediate radical(〖 I〗^•), the energy of intermediate for partial protonated PLP is 17.6 KJ/mol and for unprotonated PLP is 21.6 KJ/mol, By raising the energy of〖 I〗^•, it will increase the possibility to observe the EPR signal

In 5, 6-LAM, PLP is partially protonated because pyridine-N1 of PLP accept a hydrogen bond from Ser238, serine, even as an acid, will donate a proton to pyridine. We want to know that, first, the effect in 5, 6-LAM with electrophilicity of PLP, and second, to prove that DFT is true by comparing 1-deaza-PLP’s and mutant S238A’s reaction with 5, 6-LAM. However, the synthesis of 1-deaza-PLP hasn’t been completed, therefore we only see the comparison between 5, 6-LAM and mutant S238A.

The experiment’s result shows that the EPR spectra of 5, 6-LAM and S238A are really similar, which means that 5, 6-LAM and S238A have same structure, and still do not observe the signal of substrate radical by S238A. S238A resulted a 4.7-fold decrease in catalytic efficiency, and the rate of transaldimination was slower than 5, 6-LAM, indicating that substrate easy bond with higher protonated PLP, also affected the process include rate of homolysis and〖 Ado〗^• abstracting a hydrogen atom from the C5 of substrate-PLP complex.

第1章 緒論 1
1.1 研究背景 1
1.2 研究動機 1
1.3 研究目的與方法 2
第2章 文獻回顧 5
2.1 蛋白質簡介 5
2.2 酶簡介 5
2.2.1 輔酶 6
2.2.2 輔酶B_12─腺核苷鈷胺素 6
2.2.3 輔酶B_6─磷酸吡哆醛 8
2.3 離胺酸5,6胺基異位酶 10
2.3.1 5,6-LAM之結構 11
2.3.2 5,6-LAM之反應機制 12
2.3.3 5,6-LAM自由基中間體 13
2.3.4 5,6-LAM之磁場效應 16
2.3.5 5,6-LAM自殺效應 16
2.4 酵素動力學 17
2.4.1 Michaelis-Menten動力學模型 17
2.4.2 Lineweaver-Burk Plot 18
2.4.3 抑制作用 18
第3章 實驗儀器及原理 21
3.1 層析法 21
3.1.1 快速液相層析儀(FPLC) 21
3.1.2 陰離子交換層析 22
3.1.3 薄層層析法(TLC) 24
3.1.4 高效液相層析儀(HPLC) 24
3.2 電泳膠片分析法 25
3.3 紫外光-可見光分光光度法 27
3.4 停流反應光譜儀 28
3.5 電子順磁共振儀 30
3.5.1 離曼效應 31
3.5.2 精細結構交互作用與超精細分裂 32
3.5.3 朗德g因子 34
第4章 材料與方法 35
4.1 養菌 35
4.2 破菌 36
4.3 純化 37
4.4 TLC活性檢測 38
4.5 SDS-PAGE 分析 39
4.6 濃縮 40
4.7 酵素動力學參數測定 41
4.8 EPR測量 44
4.9 Stopped-Flow 46
第5章 結果與討論 47
5.1 純化 47
5.2 EPR光譜分析 50
5.2.1 瞬態自由基EPR訊號 50
5.2.2 穩定態自由基EPR訊號 51
5.2.3 低溫捕捉D-lysine受質自由基 52
5.2.4 Freeze Quench捕捉D-lysine受質自由基 53
5.3 HPLC酵素動力學參數實驗 54
5.4 停流反應光譜儀 60
5.4.1 Suicide Inactivation 60
5.4.2 Transaldimination 63
5.4.3 Homolysis 67
結論 69
參考文獻 71

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