Human histidine triad nucleotide binding protein 1 (hHint1) is a purine nucleoside phosphoramidase and adenylate hydrolase that has emerged as a potential therapeutic target for the management of pain. However, the molecular mechanism of Hint1 in the signaling pathway has remained less clear. The role of metal ions in regulating postsynaptic transmission is well known, and the active site of hHintl contains multiple histidines. Here we have investigated the effect of divalent metal ions (Cd2+, Cu2+, Mg2+, Mn2+, Ni2+, and Zn2+) on the structural integrity and catalytic activity of hHintl. With the exception of Mg2+, all the divalent ions inhibited hHintl, the rank of order was found to be Cu2+ >Zn2+ >Cd2+ >Ni2+ >Mn2+ based on their IC50 and k(in)/K-I values. A crystal structure of hHintl with bound Cu2+ is described to explain the competitive reversible inactivation of hHintl by divalent cations. All the metal ions exhibited time-and concentration-dependent inhibition, with the rate of inactivation highly dependent on alterations of the C-terminus. With the exception of Cu2+; restoration of inhibition was observed for all the metal ions after treatment with EDTA. Our studies reveal a loss in secondary structure and aggregation of hHintl upon incubation with 10-fold excess of copper. Thus, hHintl appears to be structurally sensitive to irreversible inactivation by copper, which may be of neurotoxicological and pharmacological significance. (C) 2017 Elsevier Inc. All rights reserved.