The role of Cu+ and ATP on the reaction cycle of a Cu+ transport ATPase from Legionella Pneumophila

PLACENTI, M. AGUEDA; ,ROMÁN, ERNESTO A.,; GONZÁLEZ FLECHA, F. LUIS; GONZÁLEZ LEBRERO, RODOLFO M.

San Luis

Congreso; XLVIII Reunion anual de la Sociedad Argentina de Biofisica; 2019

Sociedad Argentina de Biofísica

P-type ATPases form a family of membrane proteins which couple ATP hydrolysis to the transport ofsubstrates across biological membranes. Within this family, P1B-ATPases are responsible for transport oftransition metal ions, playing a key role in the regulation of their intracellular concentration. Cu+transportATPases are the most widespread and conserved members of this subfamily among different species. Inhumans, mutations of these proteins are the direct cause of Menkes and Wilson diseases. Based on somefunctional and structural evidence, it was postulated that these proteins may have a unique specificmechanism, different from that of the most studied P-ATPases.The aim of our work is to characterize the kinetics of the Cu+ transport ATPase from Legionellapneumophila (LpCopA) in order to elucidate its functional mechanism.We evaluated the role of ATP and Cu+in the reaction cycle of LpCopA by measuring steady state ATPaseactivity in presence of different concentrations of these natural ligands of the protein. We observed thatATPase activity rises as ATP concentration increases with a functional dependence that can be describedby a sum of two hyperboles. On the other hand, the increment on Cu+ concentration in the reaction mediaproduces an increment of ATPase activity that can be described by an hyperbola plus a linear function. Itis noteworthy that the ATPase activity can be measured even in the absence of Cu+.From the analysis of the effects observed we formulated a minimum kinetic model that considers: twoenzyme conformations with different affinities for ATP, enzyme phosphorylation and binding of at leasttwo Cu+ions. This model is compatible with the structural information available for this enzyme and thereaction cycle models for the most characterized P-Type ATPases.