CONICET | Buscador de Institutos y Recursos Humanos

The P5-ATPases belong to the family of ATP fuelled ion pumps which cycle between two major conformations E1 and E2 and form an acylphosphate (E~P) species as intermediate during transport. The P5-ATPases are presumed to share a catalytic mechanism similar to those of other members of the family as the Na+/K+-ATPase and the plasma membrane Ca2+-ATPase (PMCA). However, the identity of the ion which would be transported by the P5-ATPases remains elusive. In contrast there are overwhelming evidences of the importance of the P5-ATPases for the eukaryotic cell. In humans, the loss of function the ATP13A2 P5-ATPase has been shown to cause neurodegenerative diseases like Parkinsons. We have recently begun to characterize the catalytic cycle of the P5-ATPase Spf1 from Saccharomyces cerevisiae obtained by overexpression in the same yeast (Corradi et al., 2012). The purified rSpf1 is capable of hydrolyzing ATP (2.7 mmol/mg/min) and forming a phosphoenzyme in the virtual absence of metal ions with the exception of Mg2+ which is a known cofactor required for all the P-ATPases. Here we have investigated the phosphatase activity of rSpf1 by measuring the ability to hydrolyze p-nitrophenylphosphate (p-NPP). At 37 °C, in the presence of 12 mM p-NPP and 4 mM MgCl2, rSpf1 exhibited a high p-NPPase of 1.4 mmol pNOH/mg/min. This value is similar to that of other P-ATPases when stabilized in the E2 conformation. These results disagree with previous measurements of the E~P formation suggesting that the enzyme was in the E1 form (Corradi et al., 2012) and indicate that either a) a protein with pNPPase activity is present in our preparation of purified Spf1 or b) rSpf1 hydrolyzes pNPP and ATP with similar efficiency. Acknowledgments: This work is being supported by ANPCyT BID 1728 OC-AR 15-25965, y CONICET PIP 2022