臺灣博碩士論文加值系統

與受質結合後的離氨酸5,6胺基異位酶(Lysine 5,6 aminomutase, 5,6 LAM)將從開放態(Open state)進入封閉態(Close state)開始催化反應，整個反應的最初是由腺苷鈷胺中的鈷碳斷鍵，產生的第一對自由基。當進入封閉態後其中第411個胺基酸：麩胺酸，扮演一個非常重要的角色。

由於5,6 LAM的封閉態存在時間非常短，目前仍然無法直接觀察其立體結構，但在2018年，Sandeep Kumar利用生物資訊對比的方法，模擬出5,6 LAM在封閉態時的立體結構。這個結構顯示第411個胺基酸，麩胺酸官能基上的兩個氫氧根，距離腺苷鈷胺的核糖上的兩個氫氧根距離不到3Å。這個距離將顯得靜電交互作用力會非常明顯，並且認為可能會是鈷碳斷鍵的成因。

本研究將利用定點突變，將麩胺酸突變成谷氨酰胺(Glutamine)、天門冬胺酸(Aspartic acid)及丙胺酸(Alanine)，改變兩個氫氧根的距離，降低靜電交互作用力的影響，觀察其鈷碳斷鍵的能力。根據突變後的鍵長，改變靜電交互作用力的距離影響，催化能力應當是E411Q > E411D > E411A。

實驗結果顯示E411D催化能力下降約500倍，E411A及E411Q幾乎無法進行催化反應。由於丙胺酸的突變過於巨大，可能造成整個封閉態的立體結構改變，因此無法進行催化反應。E411D催化能力下降的結果顯示，鈷碳斷鍵的主要原因並不單純只是透過Glu411的影響。透過突變E411Q無法進行催化反應，但是E411D又出現活性，依照本實驗的實驗結果很難下結論去解釋其原因。未來將會朝著透過改變Adenosine上的核糖氫氧根，更直接觀察Glu411對其的影響。

After binding the substrate, lysine 5,6 aminomutase change the 3D construction from open state to close state, and start to catalyze. The beginning of the catalysis is the hemolysis of Co-C bond in adenosylcobalanine. When 5,6 LAM changed to close state, the Glu411 play an important role at the beginning of the catalysis.

As the life time of close state is very short, the 3D construction of 5,6 LAM cannot be observed directly nowadays. In 2018, Sandeep Kumar simulated the close state construction of 5,6 LAM by bio-information comparison. This result reveals that Glu411 has contribution of electrostatic energy of Co-C hemolysis. This construction shows the distance of from the hydroxide of glutamate to hydroxide on ribose of AdoCbl is nearly 3Å.

This research use site-directly mutagenisis to change Glu411 into glutamine, aspartic acid, and alanine. According to the distanece of polar side chain of amino acid, electro force will decrease E411Q > E411D > E411A. And the ability of catalysis should be E411Q > E411D > E411A.

Kcat /Km of E411D is about 500-fold decrease compare to wild type. E411A and E411Q cannot observe any activity at all. Alanine is a small amino acid. The change from glutamate to alanine is too big. And this may probabily change the whole 3D construciton of 5,6 LAM, leading protein lose activity. It is very difficult to explain why E411Q lose activity and E411D have activity. In the futrue, we’ll change adenosine into 2’-3’deoxyadenosine by chamical synthesis. It can be more directly to observe how Glu411 contribute to the adensine.

第一章 緒論 1
第二章 文獻回顧 5
第三章 實驗原理及儀器 17
第四章、實驗方法 31
第五章、實驗結果及討論 51
第六章、結論 73

1. Catilyn Makins et al. (2013). Mutagenesis of a Conserved Glutamate Reveals the Contribution of Electrostatic Energy to Adenosylcobalamin Co-C Bond Homolysis in Ornithine 4,5 Aminomutase and Methylmalonyl-CoA Mutase, Biochemistry. 52 (5), pp 878–888. doi: 10.1021/bi3012719

2. Christopher H. Chang and Perry A. Frey. (2000). Cloning, Sequencing, Heterologous Expression, Purification, and Characterization of Adenosylcobalamin-dependentD-Lysine 5,6-Aminomutase from Clostridium sticklandii. J. Biol. Chem. 2000 275: 106-. doi:10.1074/jbc.275.1.106

3. 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

4. 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

5. Kuo-Hsiang Tang, Christopher H. Chang, and Perry A. Frey (2001). Electron Transfer in the Substrate-Dependent Suicide Inactivation of Lysine 5,6-Aminomutase. Biochemistry, 2001, 40 (17), pp 5190–5199. doi: 10.1021/bi010157j