RESUMO
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.
