Steady state kinetic analysis of Legionella pneumophila Cu+ transport ATPase. The activation by Cu+ and ATP

MA. PLACENTI; RM. GONZÁLEZ LEBRERO; EA. ROMAN; FL. GONZÁLEZ FLECHA

Congreso; 20th Congress of the International Union for Pure and Applied Biophysics (IUPAB), 45th Annual SBBf Meeting, and 50th Annual SBBq Meeting; 2021

P-type ATPases are a family of membrane proteins which couple ATP hydrolysis to the transport of substrates across biological membranes. Within them, Cu+-ATPases are the most widespread and conserved heavy metal ion transporting ATPases (PIB-ATPases). Its reaction cycle is assumed to be described by the so-called Albers-Post model postulated for the most studied P-ATPases such as the Na+,K+-ATPase or the Ca2+-ATPases. However, as some structural and functional particularities arise for Cu+-ATPases, several authors posit some doubts about their reaction cycle mechanism. The aim of our work is to perform a functional characterization of Legionella pneumophila Cu⁺-ATPase (LpCopA) by measuring steady state ATPase activity. Cu+-ATPase activity of the enzyme presents a maximum at ∼37ºC and pH 6.6-6.8. Phospholipids enhance LpCopA Cu+-ATPase activity in a non-essential mode where optimal activity is achieved at an asolectin mole fraction of 0.15 and an amphiphile-protein ratio of ~30000. As described for other P-ATPases, Mg2+ acts as an essential activator. When evaluating the role of ATP and Cu+ in the reaction cycle of LpCopA we observed that ATPase activity increases as Cu+ concentration increases with a functional dependence that can be described by a sum of two hyperboles. On the other hand, the increment on ATP concentration in the reaction media produces an increment of ATPase activity that can be described by a hyperbola plus a constant value. Based on that, and the [Cu+] and [ATP] dependencies of the best fitting parameters of the functions pointed above, we propose a minimal reaction scheme for LpCopA catalytic mechanism that contemplates two enzyme conformations with different affinities for ATP, enzyme phosphorylation and binding of at least two Cu+ ions with different affinities. This model is compatible with the structural information available and the main characteristics of the reaction cycle models for the most characterized P-Type ATPases.