Impact of inter-subunit interactions on the dimeric arginine kinase activity and structural stability.
Impact of inter-subunit interactions on the dimeric arginine kinase activity and structural stability.
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DOI：
10.1016/j.abb.2011.04.015
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发表时间：
2011-08
影响因子：
3.9
通讯作者：
Qing-yun Wu;Feng Li;Xiao-yun Wang;Zheng Chen
中科院分区：
文献类型：
--
作者：
Qing-yun Wu;Feng Li;Xiao-yun Wang;Zheng Chen
精氨酸激酶（Arginine kinase， AK）是无脊椎动物细胞能量代谢的关键酶，可催化磷酸基从磷酸甘氨酸到ADP的可逆转移。本研究通过对Q53和D200之间以及D57和D200之间亚基间氢键的破坏，探讨了它们在日本刺参AK蛋白活性和结构稳定性中的作用。将Q53和/或D57突变为丙氨酸(A)会导致活性和底物协同作用的明显丧失，并引起明显的构象变化。光谱实验表明，破坏亚基间氢键的突变破坏了二聚体AK的结构，导致其部分展开。由于突变体无法折叠到功能性紧密状态，使得突变体在环境胁迫下容易失活和聚集。在Q53E和D57E突变体中恢复氢键可以挽救活性丧失和底物协同作用，以及构象变化。这些结果表明，亚基间相互作用在保持二聚体AK的活性、底物协同作用和结构稳定性方面起着关键作用。这一结果可能为理解低聚蛋白的折叠和自组装过程提供线索。
Arginine kinase (AK) is a key enzyme for cellular energy metabolism, catalyzing the reversible phosphoryl transfer from phosphoarginine to ADP in invertebrates. In this study, the inter-subunit hydrogen bonds between the Q53 and D200 and between D57 and D200 were disrupted to explore their roles in the activity and structural stability of Stichopus japonicus (S. japonicus) AK. Mutating Q53 and/or D57 to alanine (A) can cause pronounced loss of activity and substrate synergism, and cause distinct conformational changes. Spectroscopic experiments indicated that mutations destroying the inter-subunit hydrogen bonds impaired the structure of dimer AK, and resulted in a partially unfolded state. The inability to fold to the functional compact state made the mutants prone to be inactivated and aggregate under environmental stresses. Restoring hydrogen bonds in Q53E and D57E mutants could rescue the loss of activity and substrate synergism, and conformational changes. All those results suggested that the inter-subunit interactions played a key role in keeping the activity, substrate synergism and structural stability of dimer AK. The result herein may provide a clue in understanding the folding and self-assembly processes of oligomeric proteins.