We studied the Cu2+-mediated inhibition and aggregation of Exopalaemon carinicauda arginine kinase ( ECAK). We found that Cu2+ significantly inactivated ECAK activity and double-reciprocal kinetics demonstrated that Cu2+ induced noncompetitive inhibition of arginine and ATP ( IC50=2.27 +/- 0.16 mu M; K-i for arginine=13.53 +/- 3.76; K-i for ATP=4.02 +/- 0.56). Spectrofluorometry results showed that Cu2+ induced ECAK tertiary structural changes including the exposure of hydrophobic surfaces that directly induced ECAK aggregation. The addition of osmolytes such as glycine and proline successfully blocked ECAK aggregation induced by Cu2+ and recovered ECAK activity. We built a 3D structure for ECAK using the ECAK ORF gene sequence. Molecular dynamics ( MD) and docking simulations between ECAK and Cu2+ were conducted to elucidate the binding mechanisms. The results showed that Cu2+ blocked the entrance to the ATP active site; these results are consistent with the experimental result that Cu2+ induced ECAK inactivation. Since arginine kinase ( AK) plays an important role in cellular energy metabolism in invertebrates, our study can provide new information about the effect of Cu2+ on ECAK enzymatic function and unfolding, including aggregation, and the protective effects of osmolytes on ECAK folding to better understand the role of the invertebrate ECAK metabolic enzyme in marine environments.