臺灣博碩士論文加值系統

摘要
溫度是影響生物存活的主要因素之一。當變溫動物體內溫度隨環境而改變時，為了生存下去保持體內能量代謝的能力變得非常重要。鯉魚是變溫動物也是廣溫性物種，能適應35至10 oC以內的環境。本研究主要探討溫度對於酵素結構和其生理、生物特性的關係，其存在於肌肉內的肌酸激? (creatine kinase, CK)，具有維持體內能量平衡的重要作用。在pH 7.1 ~ 8.0 且0 oC ~ 20 oC 的環境下，鯉魚的肌肉型式肌酸激?，皆維持活性。而恆溫動物兔子，其肌肉內肌酸激?(RM-CK)在35 oC也維持著高活性，但在低溫下其活性會喪失，無法如鯉魚的M1型式肌酸激?(M1-CK)，在低溫下也維持著高活性。
本研究為了探討肌酸磷酸激?在低溫下結構的變化以定點突變技術，參考變溫動物的肌酸激?，改變兔子肌酸激? (RM-CK)序列268位置的Glycine 突變成 Asparagin，我們稱突變為RM-CK G268N。結果顯示RM-CK G268N在10oC且高pH值的情況下活性是RM-CK的2.5倍，且RM-CK G268N的Km值在高pH的情況下一樣維持穩定，而Kcat PCr 在15oC高pH值下略低；Kcat ADP在15oC高pH值下則相同。分別去計算其Kcat /Km PCr 、Kcat /Km ADP ，RM-CK G268N催化效率的確有較RM-CK好。熱不穩定性結果，在高pH值下，RM-CK G268N在40oC還保100%的穩定性，而RM-CK只剩60%的穩定性。RM-CK G268N 在pH 8.0，結構穩定性增加。在二級結構上，RM-CK和RM-CK G268N並沒有差異，從結晶數據來看，RM-CK G268N晶系為Tetragonal 的二聚物結構，解析度為3.3A。綜合結果推測RM-CK G268N 在與受質鍵結的區域比 RM-CK還來得緊密。在低溫且高pH的情況下，能與受質穩定的鍵結，可以幫助其提高活性。

Abstract
Temperature is a major factor that influences physiological and biochemical properties of living organisms. When ectothermic animal whose body temperature varies with the temperature of its surroundings, the regulation of energy metabolism becomes important. The eurythermic Cyprinus carpio (common Carp) can live from 35oC to 10 oC. Its muscle type creatine kinase (CK, EC 2.7.2.3) is one of the important energy metabolism enzymes which can maintain their energy requirements for survive at different temperature. There are three muscle-specific sub-isoforms of CK from Carp, designated M1-, M2- and M3-CK. The M1-CK has higher specific activity from pH7.1 ~ 8.0 and from 0 oC to 20 oC. The rabbit muscle form creatine kinase (RM-CK) exhibits its optimum activity at 35 oC. Changing the Gly 268 of RM-CK to Asn (RM-CK G268N) increased enzyme activity 2 folds at 10 oC and higher pH. The Km of RM-CK G268N remains stable as in carp M1-CK at higher pH condition. The Kcat of PCr of RM-CK G268N is lower than RM-CK at 15 oC and higher pH. The Kcat values of ADP of RM-CK G268N and RM-CK are the same at 15 oC and higher pH. Thermal stability of RM-CK G268N is higher stable at higher pH. The crystal structure of RM-CK G268N, solved at 3.3 A resolution by X-ray diffraction methods, contains one dimmer of tetragonal unit cell with dimensions a = b = 179.87 A , c = 68.74 A in the space group I4. The results show that the substrate binding site of RM-CK G268N is more compact than RM-CK. A stable substrate binding site of a two substrate enzyme is improves for activity at low temperature and high pH.

謝誌............................................................................................................. i
中文摘要...................................................................................................ii
英文摘要...................................................................................................iv
壹、 緒論..................................................................................................1
貳、 實驗方法與材料....................................................................10
一、 材料................................................................................................10
二、 方法................................................................................................14
A、建構兔子肌肉型肌酸激?定點突變...............................................14
B、純化兔子肌肉型肌酸激?和點突變體(RM-CK G268N)蛋白質…16
C、酵素活性分析.....................................................................................18
D、酵素動力學分析................................................................................18
E、熱力學穩定度分析.............................................................................19
F、圓二色光譜 Circular dichroism (CD) 分析.....................................19
G、晶體結構分析.....................................................................................20
參、 實驗結果.....................................................................................22
肆、 討論................................................................................................30
伍、 參考文獻.....................................................................................34
陸、 圖表..................................................................................................41

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