Kinetics for Cu(2+) induced Sepia pharaonis arginine kinase inactivation and aggregation.

Arginine kinase plays an important role in cellular energy metabolism and is closely related to the environmental stress response in marine invertebrates. We studied the Cu(2+)-mediated inhibition and aggregation of Sepia pharaonis arginine kinase (SPAK) and found that Cu(2+) markedly inhibited the SPAK activity along with mixed-type inhibition against the arginine substrate and noncompetitive inhibition against the ATP cofactor. Spectrofluorimetry results showed that Cu(2+) induced a tertiary structure change in SPAK, resulting in exposure of the hydrophobic surface and increased aggregation. Cu(2+)-mediated SPAK aggregation followed first-order kinetics consistent with monophasic and a biphasic processes. Addition of osmolytes, including glycine and proline, effectively blocked SPAK aggregation and restored SPAK activity. Our results demonstrated the effects of Cu(2+) on SPAK catalytic function, conformation, and aggregation, as well as the protective effects of osmolytes on SPAK folding. This study provided important insights into the role of Cu(2+) as a negative effector of the S. pharaonis metabolic enzyme AK and the possible responses of cephalopods to unfavorable environmental conditions.