ATP hydrolytic activity of purified Spf1p correlate with micellar lipid fluidity and is dependent on conserved residues in transmembrane helix M1.

P5A ATPases are expressed in the endoplasmic reticulum (ER) of all eukaryotic cells, and their disruption results in pleiotropic phenotypes related to severe ER stress. They were recently proposed to function in peptide translocation although their specificity have yet to be confirmed in reconstituted assays using the purified enzyme. A general theme for P-type ATPases is that binding and transport of substrates is coupled to hydrolysis of ATP in a conserved allosteric mechanism, however several independent reports have shown purified Spf1p to display intrinsic spontaneous ATP hydrolytic activity after purification. It has never been determined to what extend this spontaneous activity is caused by uncoupling of the enzyme. In this work we have purified a functional tagged version of the Saccharomyces cerevisiae P5A ATPase Spf1p and have observed that the intrinsic ATP hydrolytic activity of the purified and re-lipidated protein can be stimulated by specific detergents (C12E8, C12E10 and Tween20) in mixed lipid/detergent micelles in the absence of any apparent substrate. We further show that this increase in activity correlate with the reaction temperature and the anisotropic state of the mixed lipid/detergent micelles and further that this correlation relies on three highly conserved phenylalanine residues in M1. This suggests that at least part of the intrinsic ATP hydrolytic activity is allosterically coupled to movements in the TM domain in the purified preparations. It is suggested that free movement of the M1 helix represent an energetic constraint on catalysis and that this constraint likely is lost in the purified preparations resulting in protein with intrinsic spontaneous ATP hydrolytic activity. Removal of the N-terminal part of the protein apparently removes this activity.