Functioning mechanism of a Cu+ transporting ATPase from Legionella pneumophila: initial characterization

PLACENTI, M. AGUEDA; KAUFMAN, SERGIO B.; EA. ROMAN; FL. GONZÁLEZ FLECHA; RM GONZÁLEZ-LEBRERO

Congreso; XLVII Reunión Anual de la Sociedad Argentina de Biofísica.; 2018

Sociedad Argentina de Biofísica.

P-type ATPases are a family of membrane proteins which couple ATP hydrolysis tothe transport of substrates across biological membranes. Within this family, P1B-ATPases are responsible for transition metal ions transport, playing a key role inthe regulation of their intracellular concentration. Cu+ -transporting ATPases arethe most widespread and conserved members of this subfamily, being presentfrom bacteria to human, in which mutations of these proteins are the direct causeof Menkes and Wilson diseases. Even though Cu+-transporting ATPases sharefunctional and structural features with other P-type ATPases, several authorspostulate that these proteins may have a unique specific mechanism. Therefore,the aim of our work is to characterize the kinetic and thermodynamic properties ofthe Cu+ transporting ATPase from Legionella pneumophila (LpCopA) in order toelucidate its functional mechanism.LpCopA was heterologously expressed in E. Coli, solubilized in C12E10 and purifiedby affinity chromatography. We first determined the optimal conditions to measuresteady state ATPase activity of LpCopA. For that purpose, we evaluated the effectof temperature, pH, ionic strength and lipid concentration on the rate of enzymerelease of phosphate from ATP. On this basis, we then performed experiments inthe presence of different concentrations of the protein natural ligands ATP, Mg2+and Cu+. Finally, we formulated kinetic models to explain the behavioursobserved.