ABSTRACT
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 Mg(2+) (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 10(5) (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.
Subject(s)
Adenosine Triphosphatases/chemistry , Adenosine Triphosphate/chemistry , Archaeal Proteins/chemistry , Archaeoglobus fulgidus/chemistry , Copper/chemistry , Phosphoric Monoester Hydrolases/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , Archaeoglobus fulgidus/enzymology , Biocatalysis , Catalytic Domain , Cations, Divalent , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Hot Temperature , Kinetics , Magnesium/chemistry , Models, Molecular , Nitrophenols/chemistry , Organophosphorus Compounds/chemistry , Phospholipids/chemistry , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity , ThermodynamicsABSTRACT
P5-ATPases are important for processes associated with the endosomal-lysosomal system of eukaryotic cells. In humans, the loss of function of P5-ATPases causes neurodegeneration. In the yeastSaccharomyces cerevisiae, deletion of P5-ATPase Spf1p gives rise to endoplasmic reticulum stress. The reaction cycle of P5-ATPases is poorly characterized. Here, we showed that the formation of the Spf1p catalytic phosphoenzyme was fast in a reaction medium containing ATP, Mg(2+), and EGTA. Low concentrations of Ca(2+)in the phosphorylation medium decreased the rate of phosphorylation and the maximal level of phosphoenzyme. Neither Mn(2+)nor Mg(2+)had an inhibitory effect on the formation of the phosphoenzyme similar to that of Ca(2+) TheKmfor ATP in the phosphorylation reaction was â¼1 µmand did not significantly change in the presence of Ca(2+) Half-maximal phosphorylation was attained at 8 µmMg(2+), but higher concentrations partially protected from Ca(2+)inhibition. In conditions similar to those used for phosphorylation, Ca(2+)had a small effect accelerating dephosphorylation and minimally affected ATPase activity, suggesting that the formation of the phosphoenzyme was not the limiting step of the ATP hydrolytic cycle.