鯉魚肌肉型肌酸激酶在低溫下的活性取決於具極性支鏈的第268位置胺基酸

Activity of the common carp muscle form creatine kinase at low temperature depends on residue 268 with polar side chain

林希潔(Hsi-Chieh Lin)

指導教授 ： 嚴宏洋

共同指導教授 ： 許祖法(Cho-Fat Hui)

國立臺灣大學/生命科學院/漁業科學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.00039

摘要

鯉魚屬於廣溫性魚類,在35到5°C的環境中依然能維持正常的生理與代謝功能。對於鯉魚來說,如何在此廣泛的溫度下維持細胞的能量是很重要的課題。肌酸激酶是一個很重要的能量平衡酵素,可以調控並維持細胞內的能量平衡。肌肉型肌酸激酶可提供ATP給肌肉細胞以進行收縮。鯉魚具有3種同功異構的肌肉型肌酸激酶(M1-, M2- 和 M3-CK),其中只有M1-CK能夠在低溫下維持活性,而恆溫動物兔子的肌肉型肌酸激酶 (RM-CK)在低溫下則會失去活性。M1-CK和RM-CK的胺基酸序列有86%的相似性,比較兩者胺基酸的差異,以定點突變技術將RM-CK第268位置的glycine突變成asparagin,稱為RM-CK G268N。RM-CK G268N在10°C pH 8.0的情況下,活性是RM-CK的2.5倍。從這個實驗結果我們推論asparagin 268或許是M1-CK能在低溫下作用的其中一個重要原因。因此我們利用定點突變技術將M1-CK和RM-CK第268位置的胺基酸突變成aspartic acid, lysine和leucine,去研究是否是因為極性的支鏈影響其在低溫下的功能。從活性的結果來看,當268的胺基酸含有極性的支鏈時,其在低溫下的活性會比非極性的突變高,顯示極性的支鏈的確會影響M-CK低溫下的活性。從CD的實驗確認268胺基酸的改變不會影響整體的二級結構。熱不穩定性實驗則顯示含有極性268胺基酸的CK在結構上呈現比較穩定的狀態。在低溫時,水分子和水分子容易形成鍵結,造成酵素表面上的分子間親水性作用力下降,而酵素分子內的疏水性作用也降低,使得酵素的結構處於較不穩定的狀態。從結構上來看,當268胺基酸為親水性時,會和附近的其他親水性胺基酸在蛋白質表面形成一個連續的親水性區域,這個親水性區域可以增加對水分子吸引力,使水分子和親水性胺基酸形成氫鍵, 增加表面的親水性作用及分子內的疏水性作用以穩定CK的結構,因此我們認為M1-CK藉由268位置的極性胺基酸來增加和水分子之間的鍵結來穩定結構,使其可在低溫下維持活性。

關鍵字

鯉魚 ； 低溫 ； 肌肉型肌酸激酶

並列摘要

The common carp (Cyprinus carpio) required to maintain physiologic and metabolic capabilities when temperature of their habitat varies over a large temperature range 5 °C to 35 °C. At this broad range of temperature, ATP production in energy demanding cells is an important criterion to maintain the common carp. Creatine kinase is an important energy homeostasis enzyme. Muscle-specific creatine kinase (M-CK) catalyses the reversible reaction that converts phosphocreatine and ADP to creatine and ATP and thus provides ATP for muscle contraction. Previously, three common carp CK isoforms (M1-, M2- and M3-CK) were cloned. M1-CK has been suggested to have evolved to become the only CK that functions over the ranges of intracellular pH and body temperature variation that occurs in the common carp. The primary structures of RM-CK (M-CK of homeothermal rabbit) and carp M1-CK share 86% identity. By comparing the differences between RM-CK and M1-CK, and with RM-CK cDNA as template, RM-CK G268N show higher specific activity than wild type at pH 8.0 at 10 °C. This result suggests N268 residue plays an important role in conferring the appropriate 3D structure to M1-CK to function at low temperature. Therefore, we mutated 268 residues to aspartic acid, lysine or leucine and examined their properties. The mutants with polar side chain at residue 268 had higher activity and could maintain their function at low temperature. From the thermal stability, the polar mutants were more stable than non-polar mutants. Examining the structure surface of these mutants, when residue 268 was hydrophilic, it could connect nearby hydrophilic residues to form continuous hydrophilic region. This hydrophilic region could hydrogen bond with water, which then stabilizes the enzyme and results in maintaining activity at low temperature.

並列關鍵字

common carp ； low temperature ； muscle-specific creatine kinase

參考文獻

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