離胺酸5,6胺基異位酶（Lysine 5,6-Aminomutase；5,6-LAM）是一種輔酶"B" _"12" （AdoCbl）與輔酶"B" _"6" （PLP）的相依酵素。酵素5,6-LAM被發現並研究至今已有二十餘年，過去藉由受質類似物4-thia-L-lysine協助其形成穩定受質自由基的結構，以「間接」的方式來證明反應機制中存在自由基中間體；於2018年，證明5,6-LAM為磁場敏感的酵素，因催化過程涉及耦合自旋狀態的分配，「直接」證明反應機制中存在自由基中間體；因此，對於5,6-LAM的自由基連鎖反應機制，一直是個重要的問題，本篇論文將針對產物自由基（"P" ^"‧" ）的部分深入探討。
使用自然受質D-lysine的假設反應機制中，在產物自由基階段，自由基會轉移至受質六號碳上，而為了瞭解5,6-LAM於"P" ^"‧" 活性區內的相對位置，將原自然受質（D-lysine）四、五號碳間的單鍵改為雙鍵，使之成為受質類似物trans-4,5-dehydrolysine（t45DL），利用四、五號碳間的雙鍵可以穩定住六號碳上的自由基，且透過理論計算此受質類似物於"P" ^"‧" 結構時也具有較低能量，因此可能產生自由基的累計。先進行對5,6-LAM的各項條件實驗測試，在確定5,6-LAM可以接受受質類似物t45DL後，我們的長遠目標是期盼利用t45DL產生的"P" ^"‧" ，經由結構中產生自旋去區域化（spin delocalization）現象，使"P" ^"‧" 得以穩定且可以累積足夠濃度以利電子順磁共振儀（EPR）進行偵測，最後得到"P" ^"‧" 的間接證據。
純化5,6-LAM並與t45DL進行反應測試，以薄層層析（TLC）、停留光譜儀（SX20 Stopped-flow spectrometer）、快速反應設備（Freeze Quench）以及EPR協助我們了解t45DL在5,6-LAM中的生化特性，最後證實t45DL可以作為5,6-LAM的受質類似物，此外，也以理論計算結構能量之結果作為依據，透過同位素標定確定自由基位置，並確認是否可用t45DL使"P" ^"‧" 得以穩定與累積，以及進行X-band與Q-band EPR譜線的Spin Hamiltonian模擬，協助我們了解t45DL在5,6-LAM中自由基對的相對位置。

Lysine 5,6-Aminomutase (5,6-LAM) is an adenosylcobalamin (AdoCbl) and pyridoxal-5’-phosphate (PLP) dependent enzyme and follows a radical mechanism. In the past, stabilized structure of substrate radical by using substrate analogue 4-thia-L-lysine (4-S-2,6-DAH) to prove indirectly there are radical intermediates in 5,6-LAM reaction mechanism. In 2018, there is magnetic field effect in the reaction mechanism. It’s proved there are radical intermediates directly. We want to know about how does the radical reaction mechanism of 5,6-LAM work.
In the hypothetical reaction mechanism of natural substrate D-lysine, radical will be transferred to C6 of substrate when it is in the structure of product radical ("P" ^"‧" ) stage. To understand relative position in the active region, change the single bond between the C4-C5 of D-lysine to double bond, make it become a substrate analogue trans-4,5-dehydrolysine (t45DL), which uses the double bond between C4-C5 to stabilize the radical on C6. By the theoretical calculation, this substrate analogue has a lower energy in the structure of "P" ^"‧" , and it may accumulate the "P" ^"‧" . If "P" ^"‧" can be trapped and accumulate to spectroscopically detectable levels, the hypothetical mechanism of 5,6-LAM will be verified. However, we have to confirm that t45DL can be accepted by 5,6-LAM before trying to trap "P" ^"‧" .
We tested the catalytic activities of enzyme with t45DL by TLC. The biochemical characteristics and structural properties of 5,6-LAM with t45DL were investigated using Stopped-flow UV-Visible and EPR spectroscopy. We confirm that t45DL behave as a true substrate analogue for 5,6-LAM. Finally, based on the theoretical calculation of structural energy, the position of radical is determined by isotope label. And confirmed whether t45DL can be used to stabilize and accumulate "P" ^"‧" . The spin Hamiltonian simulation of X- band and Q-band EPR spectra helps us understand the relative position of the radical pairs in 5,6-LAM with t45DL.

第一章 緒論 1
1.1 研究背景 1
1.2 研究動機 1
1.3 研究目的 2
第二章 文獻回顧 3
2.1 蛋白質簡介 3
2.1.1 酵素簡介 5
2.1.2 輔酶 6
2.1.2.1 輔酶"B" _"12" 相依酵素 6
2.1.2.2 輔酶"B" _"6" 相依酵素 8
2.2 離胺酸5,6胺基異位酶簡介 9
2.2.1 5,6-LAM之結構 10
2.2.2 5,6,-LAM之輔酶 12
2.2.2.1 輔酶"B" _"12" –腺苷鈷胺 12
2.2.2.2 輔酶"B" _"6" –磷酸吡哆醛 13
2.2.3 5,6-LAM之假設反應機制 15
2.2.4 5,6-LAM之自由基中間體 17
第三章 實驗儀器與原理 23
3.1 色層分析法 23
3.1.1 快速液相層析儀（FPLC） 23
3.1.1.1 疏水層析法 24
3.1.1.2 陰離子交換層析法 25
3.1.2 薄層層析法（TLC） 27
3.2 電泳膠片分析法 28
3.3 紫外光-可見光分光光度計 30
3.4 停流反應光譜儀 31
3.5 Freeze Quench（System 1000） 32
3.6 電子順磁共振儀 34
3.6.1 塞曼效應（Zeeman effect） 34
3.6.2 精細結構交互作用（Hyperfine Interaction） 36
3.6.3 電子-電子交互作用（Electron-Electron Interaction） 37
3.6.4 X-band與Q-band EPR 39
3.6.5 朗德g因子 40
第四章 材料與方法 41
4.1 養菌 41
4.2 破菌 42
4.3 純化 43
4.4 TLC活性檢測 46
4.5 SDS-PAGE分析 47
4.6 Stopped-flow UV-Visible光譜 49
4.7 Freeze Quench 50
4.8 EPR量測 51
第五章 結果與討論 53
5.1 5,6-LAM蛋白質純化 53
5.1.1 Phyenl疏水性層析 53
5.1.1.1 TLC活性測試（Phyenl） 53
5.1.1.2 SDS-PAGE測試（Phyenl） 54
5.1.2 QFF陰離子交換層析 54
5.1.2.1 TLC測試（QFF） 55
5.1.2.2 SDS-PAGE測試（QFF） 55
5.2 TLC活性分析 56
5.3 Stopped-flow UV-Visible光譜分析 57
5.4 EPR光譜分析 58
5.4.1 X-band EPR 58
5.4.2 Q-band EPR 65
5.5 討論 68
第六章 結論與總結 71
參考文獻 73

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