肌酸激酶（CK，adenosine-5’-triphosphate:creatine phosphotransferase，EC 2.7.3.2）催化磷酸肌酸和ADP之间可逆的转磷酰基反应，生成肌酸和ATP。肌酸激酶是一种古老的磷酸原激酶，在十几亿年的进化过程中从单一祖先蛋白形成了具有不同亚细胞定位和组织分布的同工酶家族，成为脊椎动物体内唯一的磷酸原激酶。本论文主要研究了两种人的胞浆型肌酸激酶（脑型和肌肉型）由于面临不同的体内微环境压力，表现出同工酶特异性的热稳定性，并发现36位氨基酸是造成这种热稳定性差异的关键氨基酸；随后我们从进化角度研究了肌肉型肌酸激酶的高热稳定性是如何形成的并发现46位氨基酸在肌肉型肌酸激酶热稳定性进化过程中起关键作用。人肌肉型肌酸激酶（hMMCK）和人脑型肌酸激酶（hBBCK）的热稳定性有很大差异，两者的半失活温度分别为56.5°C和41.8°C。hMMCK的热失活过程是两态不可逆过程，并伴随着大量聚沉产生，而hBBCK的热失活使部分可逆的，而且没有明显聚沉产生。分子筛层析发现在hBBCK热失活过程中有二聚体解聚的发生并且单体hBCK可以重新聚合为二聚体。但是分子筛层析没有检测到单体hMCK的存在，说明其性质极不稳定，一旦产生马上聚沉。通过结构域置换的方法，我们找到了27-53氨基酸是影响热稳定性的关键亚结构域，而进一步的点突变研究表明36位氨基酸是影响热稳定性的关键氨基酸，并且通过影响亚基间相互作用强度影响热稳定性。这一结果说明了二聚体解聚是肌酸激酶热失活过程中的关键步骤，而36位氨基酸在区分同工酶特异性热稳定性中发挥关键作用。我们进一步检测了MMCK热稳定性的进化过程，通过比对34种MMCK我们发现有9个位点在同一纲动物中具有保守性而在不同纲动物中则大不相同。通过比较这9个位点突变对人MMCK和斑马鱼MMCK的热稳定性的影响，我们发现46位氨基酸是进化过程中MMCK热稳定性提高的关键氨基酸，而146位和329位氨基酸则在鱼类MMCK适应水生环境过程中发挥关键作用。

Creatine kinase (CK, EC 2.7.3.2) catalyzes the reversible transfer of a phosphoryl group from phosphocreatine to ADP, producing creatine and ATP. CK is one of the most ancient members of phosphagen kinase superfamily. CK evolved a large family of isoenzymes with different intracellular location and tissue distribution from one ancestor protein in more than one billion years and becomes the only phosphagen kinase in vertebrates. In this thesis, we found the isoenzyme-specific thermostabilities of two human cytosolic CK (hMMCK and hBBCK) caused by their different tissue distributions and identified the 36th residue as the key amino acid residue in differentiating the thermostability of hBBCK from hMMCK. We next investigated the evolutionary formation of the higher thermostability of MMCK and found the 46th residue played a key role in the evolution of MMCK thermostability.The semi-inactivation temperatures of hMMCK and hBBCK are 41.8°C and 56.5°C separately. Thermal inactivation of hMMCK is an irreversible process accompanied with severe aggregation, while thermal inactivation of hBBCK is a partially reversible process with no apparent aggregation. Gel filtration showed the dimer dissociation and the re-association of the monomeric hBCK was the reason for inactivation and reactivation of hBBCK, but no monomeric hMCK was detected, which was consistent with its irreversibility. Through domain-swapping, the region from the 27th to 53rd amino acid residue was identified as the decisive region for thermal stability. Further sequence scanning and site-directed mutation showed the 36th amino acid was the key residue for thermal stability of CK, and gel filtration showed the mutation influenced the strength of dimer cohesion and led to the change of thermal stability. The results herein indicated that dimer dissociation was the key step in CK thermal inactivation, and the 36th amino acid residue played an important role in the isoform-specific stability of human CKs, which is proposed to be associated with the tissue-specific physiological conditions. We further detected the evolutionary formation of the higher thermostability of MMCK。Through sequence alignment of 34 MMCKs, we found 9 amino acids were the conserved within classes but very different among classes. Then we investigated the influence of mutagenesis on these 9 position and finally identified the 46th residues as the key amino acid in the thermostability evolution of MMCK, and the 146th and 329th play an important role in the adaptation to aquatic environment of teleosts.