The promiscuous phosphomonoestearase activity of Archaeoglobus fulgidus CopA, a thermophilic Cu+ transport ATPase

• Autores: Bredeston, Luis María; Gonzalez Flecha, Francisco Luis
• Año de publicación: 2016
• Idioma: inglés
• Tipo de recurso: artículo
• Materia: Membrane Proteins; Enzyme Catalysis; P-Atpase; Principal Activity; Catalytic Proficiency; Enzyme Promiscuity
• Institución: Consejo Nacional de Investigaciones Científicas y Técnicas

Membrane transport P-type ATPases display two characteristic enzymatic activities: a principal ATPase activity provides the driving force for ion transport across biological membranes, whereas a promiscuous secondary activity catalyzes the hydrolysis of phosphate monoesters. This last activity is usually denoted as the phosphatase activity of P-ATPases. In the present study, we characterize the phosphatase activity of the Cu+-transport ATPase from Archaeglobus fulgidus (Af-CopA) and compare it with the principal ATPase activity. Our results show that the phosphatase turnover number was 20 times higher than that corresponding to the ATPase activity, but it is compensated by a high value of Km, producing a less efficient catalysis for pNPP. This secondary activity is enhanced by Mg2 + (essential activator) and phospholipids (non-essential activator), and inhibited by salts and Cu+. Transition state analysis of the catalyzed and noncatalyzed hydrolysis of pNPP indicates that Af-CopA enhances the reaction rates by a factor of 105 (ΔΔG‡ = 38 kJ/mol) mainly by reducing the enthalpy of activation (ΔΔH‡ = 30 kJ/mol), whereas the entropy of activation is less negative on the enzyme than in solution. For the ATPase activity, the decrease in the enthalpic component of the barrier is higher (ΔΔH‡ = 39 kJ/mol) and the entropic component is small on both the enzyme and in solution. These results suggest that different mechanisms are involved in the transference of the phosphoryl group of p-nitrophenyl phosphate and ATP.
Fil: Bredeston, Luis María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; Argentina
Fil: Gonzalez Flecha, Francisco Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; Argentina