CONICET | Buscador de Institutos y Recursos Humanos

P-type ATPases form a family of membrane proteins which couple ATP hydrolysis to thetransport of substrates across biological membranes. Within this family, P1B-ATPases areresponsible for transport of transition metal ions, playing a key role in the regulation oftheir intracellular concentration. Cu+ transport ATPases are the most widespread andconserved members of this subfamily among different species. In humans, mutations ofthese proteins are the direct cause of Menkes and Wilson diseases. Based on somefunctional and structural evidence, it was postulated that these proteins may have aunique specific mechanism, different from that of the most studied P-ATPases.The aim of our work is to characterize the kinetics of the Cu+ transport ATPase fromLegionella pneumophila (LpCopA) in order to elucidate its functional mechanism.We evaluated the role of ATP and Cu+ in the reaction cycle of LpCopA by measuringsteady state ATPase activity in presence of different concentrations of these naturalligands of the protein. We observed that ATPase activity rises as ATP concentrationincreases with a functional dependence that can be described by a sum of twohyperboles. On the other hand, the increment on Cu+ concentration in the reactionmedia produces an increment of ATPase activity that can be described by an hyperbolaplus a linear function. It is noteworthy that the ATPase activity can be measured even inthe absence of Cu+ .From the analysis of the effects observed we formulated a minimum kinetic model thatconsiders: two enzyme conformations with different affinities for ATP, enzymephosphorylation and binding of at least two Cu+ ions. This model is compatible with thestructural information available for this enzyme and the reaction cycle models for themost characterized P-Type ATPases