低溫下鯉魚肌肉型肌酸激酶之生化及結構研究

Abstract

全球氣候變遷是生物所需面對的一個重要課題。在台灣，每年寒流所造成水產養殖上的經濟損失高達數千萬元以上。魚類生物學家基於魚類逆境生理的分子機制研究，發展出各種技術來降低經濟損失。在此必須先了解硬骨魚類為了應付溫度的變化，發展出避免周遭溫度的傷害的各種生理生化機制。 由於鯉魚可以生存在35 到5 °C之間，之前研究發現鯉魚肌肉型肌酸激酶可以在低溫有較佳的活性，為了了解肌肉型肌酸激酶的分子機制，我們取兩種鯉魚肌肉型肌酸激酶(M1-, M3-CK)和兔子的肌肉型肌酸激酶(RM-CK)來作比較。發現在15 °C , pH 7.7以上時，M1-CK可以比M3-CK和RM-CK多3-8倍的活性。而且，M1-CK在pH 8.0時，在15 °C的狀況下具有最高活性，同時M1-CK 的酵素動力學特性KmPCr 和 KmADP，在不同溫度和pH值下，相對穩定。其催化反應的活化能(Ea)也比較低。從圓二色光譜也發現，M1-CK在不同的測試溫度和pH值下也都維持不變。 當我們將RM-CK第268個胺基酸，甘胺酸，用M1-CK同位置的天冬醯胺酸來取代後，RM-CK G286N的變異蛋白在10 °C, pH 8.0的狀況下，活性為野生型RM-CK的2倍。動力學特性上，兩者的Km並沒有太大的差異然而圓二色光譜卻發現，RM-CK G286N在5 °C, pH 8.0的狀況下和鯉魚的M1-CK極為相似。RM-CK G286N結晶的X光繞射圖譜解出其受質結合區域的胺基酸互相靠近，反應中心的胱胺酸283則從ADP的結合部位向外突出。在pH 7.4-8.0之間，用體積較小的dADP當受質的話，RM-CK G268N會有較高的反應活性，類似M1-CK。 接著，我們把RM-CK和M1-CK的第268位置的胺基酸分別突變為天冬胺酸，離胺酸和白胺酸，用以研究這個位置對整個酵素的生物物化特性的影響。其中天冬胺酸和離胺酸變異的肌酸激酶則會有和天冬醯胺酸突變的酵素一樣具有低溫活性，而白胺酸變異的肌酸激酶則沒有。在進行催化反應的緩衝液中加入甘油，可以發現親水性的側鏈有助於穩定酵素低溫下的活性。 綜上所述，我們認為鯉魚M1-CK演化出適應低溫環境的功能，從RM-CK G268N結構分析發現，其反應中心空間縮小為其具有低溫活性的原因，從動力學和疏水性環境的試驗中得知，第268個胺基酸側鏈和水分子的作用會影響酵素的構形，減少該側鏈的疏水性也會同時降低酵素的不穩定性而維持其反應活性。基於這個研究結果，希望可以解開酵素如何在低溫下維持其反應活性的原因。

Extreme environmental change is an immediately challenge all over the world. The cold fronts sweeping in the winter, which causes millions of losses in aquaculture, is a severe challenge of Taiwan aquaculture industry. Marine biologists have developed some techniques to minimize the economic loss based on the studies of molecular mechanism of fish in stress. To overcome the change of temperature, physiologically, teleost has developed lots of mechanism to avoid harmful damage of ambient environment. The physiological effects of low temperature have mainly focused on following issues: metabolic compensation, homeoviscous adaptation of biological membranes, and thermal hysteresis. The common carp could live from 35 to 5 °C. Its muscle-specific creatine kinase (M-CK) could maintain enzymatic activity at temperature around 15 °C. The present studies focus on the three common carp M-CK sub-isoforms (M1-, M2- and M3-CK) which are important in energy homeostasis. Specific activities of the common carp M1-CK were 3 to 8-folds higher than specific activities of M3- and rabbit M-CK at temperatures below 15 °C and pHs above 7.7. KmPCr and KmADP of M1-CK were relatively stable at pHs between 7.1 to 8.0, 25 to 5 °C. Its calculated activation energy of catalysis (Ea) at pH 8.0 was lower than at pH 7.1. Circular dichroism spectroscopy results showed that changes in secondary structures of M1-CK at the pHs and temperatures under studied were much less than in the cases of rabbit muscle-specific creatine kinase (RM-CK) and M3-CK. When glycine 268 in RM-CK was substituted with asparagine 268 as found in carp M1-CK, the RM-CK G286N mutant specific activity at pH 8.0, 10 °C was more than 2-fold higher than the wild-type RM-CK at the same condition. Kinetic studies showed that Km values of the RM-CK G268N mutant were similar to those of the RM-CK, yet circular dichroism spectrum showed that the overall secondary structures of the RM-CK G268N, at pH 8.0, 5 °C, was almost identical to the carp M1-CK enzyme. The X-ray crystal structure of the RM-CK G268N revealed that amino acid residues involved in substrate binding were closer to one another than in the native RM-CK, and the side chain of cysteine 283 in active site of the RM-CK G268N pointed away from the ADP binding site. At pH 7.4-8.0, 35-10 °C, with a smaller substrate, dADP, specific activities of the mutant enzyme were consistently higher than the RM-CK and more similar to the carp M1-CK. Then, to study the changes in physico-biochemical properties caused by residue 268 in RM-CK and M1-CK at low temperature, six more mutants, aspartic acid 268, lysine 268 or leucine 268 of RM-CK and M1-CK were generated. The peptide fragments near the active site were found to be phosphorylated. The specific activity results showed that, as in the case of asparagine 268, the aspartic acid 268 and lysine 268 mutants exhibited higher specific activities at low temperature and at higher pH, but not the leucine 268 mutant. The lower hydrophobicity side chain of residue 268 may help the stability of enzyme in glycerol containing buffer. To sum up, we have found out that, the M1-CK enzyme seems to have evolved to adapt to the synchronized changes in body temperature and intracellular pH of the common carp. The smaller active site of the RM-CK G268N mutant might be one of the reasons for M-CK to improve activity at low temperature. The kinetic results and glycerol influence results indicated that charged side chain of residue 268 of M-CK might cause changes in protein conformation by interacting with water, and decreasing hydrophobicity of M-CK which in turn decreased its instability at low temperature.