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

P-type ATPases are a family of membrane proteins which couple ATPhydrolysis to the transport of substrates across biological membranes. Withinthem, Cu + -ATPases are the most widespread and conserved heavy metal iontransporting ATPases (PIB-ATPases). Its reaction cycle is assumed to bedescribed by the so-called Albers-Post model postulated for the most studied P-ATPases such as the Na + ,K + -ATPase or the Ca 2+ -ATPases. However, as somestructural and functional particularities arise for Cu + -ATPases, several authorsposit some doubts about their reaction cycle mechanism. The aim of our work isto perform a functional characterization of Legionella pneumophila Cu⁺-ATPase(LpCopA) by measuring steady state ATPase activity. Cu + -ATPase activity of theenzyme presents a maximum at ∼37ºC and pH 6.6-6.8. Phospholipids enhanceLpCopA Cu + -ATPase activity in a non-essential mode where optimal activity isachieved at an asolectin mole fraction of 0.15 and an amphiphile-protein ratio of~30000. As described for other P-ATPases, Mg 2+ acts as an essential activator.When evaluating the role of ATP and Cu + in the reaction cycle of LpCopA weobserved that ATPase activity increases as Cu + concentration increases with afunctional dependence that can be described by a sum of two hyperboles. Onthe other hand, the increment on ATP concentration in the reaction mediaproduces an increment of ATPase activity that can be described by a hyperbolaplus a constant value. Based on that, and the [Cu + ] and [ATP] dependencies ofthe best fitting parameters of the functions pointed above, we propose aminimal reaction scheme for LpCopA catalytic mechanism that contemplatestwo enzyme conformations with different affinities for ATP, enzymephosphorylation and binding of at least two Cu + ions with different affinities. Thismodel is compatible with the structural information available and the maincharacteristics of the reaction cycle models for the most characterized P-TypeATPases.Fil: Placenti, Maria Agueda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Roman, Ernesto Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Gonzalez Flecha, Francisco Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Gonzalez-Lebrero, Rodolfo Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina20th Internaitional Congress of the INternational Union for Pure Applied Biophysics; 50th Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology; 45th Congress of Brazilian Biophysics Society anda 13th Brazilian Society on Nuclear Biosciences CongressBrasilSociedade Brasileira de Bioquímica e Biología Molecula

Functional characterization of Legionella pneumophila Cu+ transport ATPase: The activation by Cu+ and ATP

Cu+-ATPases are integral membrane proteins belonging to the IB subfamily of the P-type ATPases that couple Cu+ transport to the hydrolysis of ATP. As some structural and functional particularities arise for Cu+-ATPases, several authors suggest that some of the reaction steps of the Albers-Post model postulated for other P-ATPases may be different. In this work we describe a functional characterization of Legionella pneumophila Cu⁺-ATPase (LpCopA), the first PIB-ATPase whose structure was determined by X-ray crystallography. 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 molar fraction of 0.15 and an amphiphile-protein ratio of ~30000. As described for other P-ATPases, Mg2+ acts as an essential activator. Furthermore, Cu+-ATPase activity dependence on [Cu+] and [ATP] can both be described by a sum of two hyperbolic functions. Based on that, and the [Cu+] and [ATP] dependencies of the best fitting parameters of the hyperbolae pointed above, we propose a minimal reaction scheme for the catalytic mechanism that shares the basic reaction steps of the Albers-Post model for P-type ATPases. The reaction scheme postulated contemplates two different binding affinities for a single ATP (apparent affinities of 0.66 and 550 μM at [Cu+]→∞) and, binding of at least, 2 Cu+ with different affinities as well (apparent affinities of 1.4 and 102.5 μM at [ATP]→∞).Fil: Placenti, Maria Agueda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Roman, Ernesto Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Gonzalez Flecha, Francisco Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Gonzalez-Lebrero, Rodolfo Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin

Unexpected Effects of K + and Adenosine Triphosphate on the Thermal Stability of Na + ,K + -ATPase

Na+,K+-ATPase is an integral membrane protein which couples ATP hydrolysis to the transport of three Na+ out and two K+ into the cell. The aim of this work is to characterize the effect of K+, ATP, and Mg2+ (essential activator) on the Na+,K+-ATPase thermal stability. Under all conditions tested, thermal inactivation of the enzyme is concomitant with a structural change involving the ATP binding site and membrane-associated regions. Both ligands exert a clear stabilizing effect due to both enthalpic and entropic contributions. Competition experiments between ATP and K+ showed that, when ATP is present, the inactivation rate coefficient exhibits a biphasic dependence on K+ concentration. At low [K+], destabilization of the enzyme is observed, while stabilization occurred at larger cation concentrations. This is not expected for a simple competition between the enzyme and two ligands that individually protect the enzyme. A model that includes enzyme species with none, one, or two K+ and/or one molecule of ATP bound explains the experimental data. We concluded that, despite both ligands stabilizing the enzyme, the species with one K+ and one ATP simultaneously bound is unstable.Fil: Placenti, Maria Agueda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; ArgentinaFil: Kaufman, Sergio Benjamín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; ArgentinaFil: Gonzalez Flecha, Francisco Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; ArgentinaFil: Gonzalez-Lebrero, Rodolfo Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; Argentin