The promiscuous phosphomonoestearase activity of Archaeoglobus fulgidus CopA a thermophilic Cu+ transport P-type ATPase

LUIS M. BREDESTON; F. LUIS GONZÁLEZ FLECHA

BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2016 vol. 1858 p. 1471 - 1478

0005-2736

Membrane transport P-type ATPases display two characteristic enzymaticactivities: a principal ATPase activity provides the driving force for iontransport across biological membranes, whereas a promiscuous secondary activitycatalyzes the hydrolysis of phosphate monoesters. This last activity is usuallydenoted as the phosphatase activity of P-ATPases. In the present study wecharacterize the phosphatase activity of the Cu+-transport ATPase fromArchaeglobus fulgidus (Af-CopA) and compare it with theprincipal ATPase activity. Our results show that the phosphatase turnovernumber was 20 times higher than that corresponding to the ATPase activity, butit is compensated by a high value of Km,producing a less efficient catalysis for pNPP. This secondary activity isenhanced by Mg2+ (essential activator) and phospholipids(non-essential activator), and inhibited by salts and Cu+. Transitionstate analysis of the catalyzed and non catalyzed hydrolysis of pNPP indicatesthat Af-CopA enhances the reactionrates by a factor of 105 (ΔΔG? = 38 kJ/mol) mainly byreducing the enthalpy of activation (ΔΔH? = 30 kJ/mol) whereas theentropy of activation is less negative on the enzyme than in solution. For theATPase activity the decrease in the enthalpic component of the barrier ishigher (ΔΔH? = 39 kJ/mol) and the entropic component is small on boththe enzyme and in solution. These results suggest that different mechanisms areinvolved in the transference of the phosphoryl group of p-nitrophenyl phosphateand ATP.