Hydrolysis of nucleotide and non-nucleotide substrates by the Spf1p P5A-ATPase

ARPI JA; CORRADI, GERARDO R.; MAZZITELLI L.R.; ADAMO HP

San Pablo

Congreso; 20th International Congress of IUPAB a joint meeting with the 45th. Brazilian Congress of SBBf and 50th Annual Meeting of SBBq; 2021

P-ATPases are integral membrane proteins that couple the hydrolisis of ATP with the transport of ions and lipids against their concentration gradient.The first molecular structure was obtained in 2000 and showed that P-ATPases have 3 citoplasmic domains:N domain, which bind the ATP moleculeP domain, with a conserved aspartate residue that is phosphorylated during the transport cicleand A domain, with a conserved TGES sequence that removes phospate from the protein.All P type ATPases share a coomon reaction cycle, briefly they alternate between two conformational states E1 and E2.E1 binds ATP and in the presence of the subtrate being transported forms the high energy phosphoenzyme E1P. Concomitant with the ion trasnport E1P is converted to the low energy phosphoenzyme E2P. then the TGES motive of A domain promotes the released of Pi while the counter ion transport takes place.ATP also binds to a regulatory site on the enzyme and high concentrations of ATP in the reaction media accelerates the rate of the reaction cicle. Besides, Several Works showed that membrane and purified preparations of some ATPases (such as SERCA but not PMCA) can hydrolize nucleotides different from ATP and with some of them could support ion transport.P-ATPAases are divided into 5 submafilies. The P5 subfamily is the less characterized. They were subdivided in two groups: P5A and P5B.In this study I will present resaults corresponding to the yeast P5A-ATPase Spf1. Deletion of Spf1 in yeast has a pleiotropic effect, but mainly causes ER stress and triggers the UPR. Also deletion of the homologous protein in C elegans alters dendrite formation in some neuron. Last year the function of this protein was discovered. An article published in Science jounal found that Spf1 translocate membrane peptide of protein misinserted in the ER membrane. In the case of Spf1, the subtrate are not ion or lipid but membrane peptides and put Spf1 within the proteoatasis control network.Ø Well, the aim of this study is the Functional characterization of P5-ATPases using Spf1p as a model. The idea is to characterize its active site and to address the ability of Spf1 to use other phosphorilated compounds as substrate.We used purified preparation of Spf1 in detergent miscelles. and perform the enzimatic assay in a simple reaction media containing only buffer, and MgThe first result shows that Spf1p can hydrolyze ITP and GTP at a similar rate than ATP but other nucleotides are less hydrolyzed. To study with more detail how was the hydrolisis of ITP and UTP we measured the rate of hydrolysis as a funcion of nucleotide concentration in a range from 50 uM to 3 mM (so we are not measuring the catltyc site which affinity in under 10 uM). The was similar for all nucleotides assayed but Km was higher for ITP and UTP than ATP suggesting that the acceleration of the reaction cycle by these nucleotides is impared.Some ATPase can also hydrolyze non nucleotidic compounds although the importance of this activity is not clear. But this ability has been used to characterize the substrate binding site.In this regard, we tested if Spf1 was able to hydrolyze PP or pNPP and only pNPP hydrolysis was supported by Spf1. Again we performed a rate of hydrolysis vs substrate cc (in this case pNPP) and we could see that the best fit to experimental data was an hyperbole with an origin suggestin that also pNPP has a high affinity binding site on Spf1p. Some P-ATPases may sustain pNPP hydrolysis uncoupled from the normal transport reaction cycle but it seems that this is not the case of Spf1p. Also the rate of hydrolysis of pNPP was 4 times higher than that of ATP