Leishmania amazonensis: inhibition of 3'-nucleotidase activity by Cu2+ ions

Free Cu(2+) is toxic due to the capacity of free copper ions to catalyze the production of reactive oxygen species (ROS) that can modify the structure and/or function of biomolecules. In addition, non-specific binding to enzymes, which modifies their catalytic activities, can occur. In this work, the mechanisms underlying the ability of copper to inhibit 3'-nucleotidase from Leishmania amazonensis (La3'-nucleotidase) were investigated. To that end, La3'-nucleotidase activity was assayed with CuCl(2) in the presence of ascorbate or hydrogen peroxide to discriminate non-specific binding effects from pro-oxidant effects of copper. Copper inhibitory effects were greater at more acidic pH than at alkaline pH. The addition of enzyme substrate, adenosine 3'-monophosphate (3'AMP), prevented the inhibition of enzyme activity by copper. Thiol-containing compounds were able to protect the enzyme activity against inhibition due to copper. The specific copper chelating agent bathocuproine sulphonate (BCS) restored enzyme activity after pre-treatment of the enzyme with copper. La3'-nucleotidase activity was found to be resistant to ROS generated during oxidation reactions of ascorbate and hydrogen peroxide catalyzed by copper. Our results suggest that Cu(2+) ions exert their inhibitory effects by binding to specific motifs of the 3'-nucleotidase protein and that the enzyme appears to be extremely resistant to ROS.