Temperature and pH dependence of Cu+ ATPase activity of LpCopA

BATASTINI, N.A.; MA. PLACENTI; EA. ROMAN; RM GONZALEZ LEBRERO

Congreso; LI Reunión Anual de la Sociedad Argentina de Biofísica; 2023

P-ATPases are a family of membrane proteins which couple ATP hydrolysis to the transport of substrates across biological membranes. Within the P-ATPases family, P1B-ATPases are responsible for transition metal ions transport, where Copper transporting ATPases (CopA) are the most conserved and widely spread. Even though they share structural and functional elements with other P-ATPases, CopAs show distinctive features. The aim of the work presented here is to characterize the effect of temperature and pH on the functionality of a Cu+ transporter of Legionella pneumophila (LpCopA), a mesophilic bacteria.For that, we measured steady state ATPase activity as a function of [ATP] at different temperatures (from 17ºC to 42ºC) and pHs (from 6 to 7). Our results were well described by a hyperbola plus a constant term at all temperatures and pHs tested. The values of the apparent dissociation constant of the enzyme-ATP complex (KM), the Catalytic Efficiency (kcat/KM), and the catalytic constant (kcat) were obtained. Both kcat and kcat/KM increase with temperature, reaching a maximum (at 34ºC for kcat/ KM, 37ºC for kcat) and then decrease. Conversely, KM was constant until 30ºC, after which it increased exponentially as a function of temperature. Both dependencies can be described considering a reversible inactivation of LpCopA. The thermodynamic activation parameters values indicated that the free energy barrier (ΔG‡) is lowered by an enthalpy change. On the other hand, kcat increases with pH up to a maximum at pH 6.6 and then decreases suggesting at least two protonation events with opposite effects. Considering a Hill Model for proton binding, the protonation responsible for the activation event involves at least 4 H+ with an apparent pKa of 6.27±0.04, while the protonation responsible for the inhibition event involves at least 2 H+ with an apparent pKa of 7.0±0.7. KM was constant at 272 ± 89 μM until pH 6.6, increasing to >5000 μM at higher pHs.AcknowldegmentsWith grants from UBA, CONICET and ANPCyT