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

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

La Plata, Buenos Aires, Argentina

Congreso; XLVII Reunion anual de la Sociedad Argentina de Biofisica; 2018

Sociedad Argentina de Biofísica

P-typeATPases are a family of membraneproteinswhich couple ATP hydrolysis to the transport of substrates acrossbiological membranes. Within this family, P1B-ATPases are responsiblefor transition metal ions transport, playing a key role in theregulation of theirintracellular concentration. Cu+-transportingATPases are the most widespread and conserved members of thissubfamily, being present from bacteria to human, in which mutationsof these proteins arethe direct causeofMenkesand Wilson diseases. Even though Cu+-transportingATPases share functional and structural features with other P-typeATPases,several authors postulate that theseproteins may have a unique specific mechanism. Therefore,the aimof ourwork is to characterize the kinetic and thermodynamic properties ofthe Cu⁺transporting ATPase from Legionellapneumophila (LpCopA)in order toelucidate its functionalmechanism.LpCopAwas heterologously expressed in E.Coli,solubilized in C12E10and purified by affinity chromatography. Wefirstdeterminedthe optimal conditions to measure steady state ATPase activity ofLpCopA.Forthat purpose, weevaluatedthe effect of temperature, pH, ionic strength and lipid concentrationonthe rateof enzymereleaseof phosphate from ATP.Onthis basis, we thenperformedexperiments in the presence of different concentrations of theprotein natural ligands ATP, Mg2+and Cu+.Finally,we formulated kinetic models to explain the behaviours observed.p { margin-bottom: 0.25cm; line-height: 120%; }