Impact of inter-subunit interactions on the dimeric arginine kinase activity and structural stability

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. (c) 2011 Elsevier Inc. All rights reserved.