Arginine kinase plays an important role in invertebrate cellular energy metabolism. We investigated the effects of Cu2+ on the enzymatic activity, unfolding and aggregation of Euphausia superba arginine kinase (ESAK). Cu2+ inhibited the activity of ESAK (IC50 = 0.050 +/- 0.002 mM) following first-order kinetics consistent with the transition from a monophasic to a biphasic reaction. Double-reciprocal Lineweaver-Burk plots indicated that Cu2+ induced non-competitive inhibition of arginine and ATP. Spectrofluorimetry results showed that Cu2+ induced tertiary structural changes in ESAK with exposure of hydrophobic surfaces and directly induced ESAK aggregation. The addition of osmolytes such as glycine and praline successfully blocked ESAK aggregation, recovering the conformation and activity of ESAK. Computational simulations indicated that the ATP/ADP binding site locations overlap with the Cu2+ interacting residues, inferring that Cu2+ binding can block ESAK enzyme catalysis with ATP/ADP cofactor binding via modulating structural conformation. Our study demonstrates the effect of Cu2+ on ESAK enzymatic function and folding mechanisms that might provide important insights into other metabolic enzymes of invertebrates in extreme climatic marine environments. (C) 2014 Elsevier Ltd. All rights reserved.