title: Leishmania amazonensis: inhibition of 3'-nucleotidase activity by Cu2+ ions.
abstract: 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.
authors.affiliation: Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, CCS, Bloco H, Cidade Universitária, Ilha do Fundão, 21941-590 Rio de Janeiro, Brazil.
authors.forename: Rafael
authors.initials: R
authors.lastname: Paletta-Silva
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Vieira-Bernardo
authors.forename: Daniela
authors.initials: D
authors.lastname: Cosentino-Gomes
authors.forename: José Roberto
authors.initials: JR
authors.lastname: Meyer-Fernandes
mesh_terms: D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism; D003300:Copper / Q000378:metabolism / Q000494:pharmacology*; D006224:Cricetinae; D003545:Cysteine / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005978:Glutathione / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007894:Leishmania mexicana / Q000187:drug effects / Q000201:enzymology*; D008623:Mercaptoethanol / Q000494:pharmacology; D009708:Nucleotidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010618:Phenanthrolines / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D010618:Phenanthrolines; D017382:Reactive Oxygen Species; D008623:Mercaptoethanol; C028559:bathocuproine sulfonate; D003300:Copper; D006861:Hydrogen Peroxide; D009708:Nucleotidases; C040470:3'-nucleotidase; D005978:Glutathione; D003545:Cysteine; D001205:Ascorbic Acid; C029892:cupric chloride; D010100:Oxygen; D004229:Dithiothreitol