title: Metal Fluoride Inhibition of a P-type H+ Pump: STABILIZATION OF THE PHOSPHOENZYME INTERMEDIATE CONTRIBUTES TO POST-TRANSLATIONAL PUMP ACTIVATION.
abstract: The plasma membrane H(+)-ATPase is a P-type ATPase responsible for establishing electrochemical gradients across the plasma membrane in fungi and plants. This essential proton pump exists in two activity states: an autoinhibited basal state with a low turnover rate and a low H(+)/ATP coupling ratio and an activated state in which ATP hydrolysis is tightly coupled to proton transport. Here we characterize metal fluorides as inhibitors of the fungal enzyme in both states. In contrast to findings for other P-type ATPases, inhibition of the plasma membrane H(+)-ATPase by metal fluorides was partly reversible, and the stability of the inhibition varied with the activation state. Thus, the stability of the ATPase inhibitor complex decreased significantly when the pump transitioned from the activated to the basal state, particularly when using beryllium fluoride, which mimics the bound phosphate in the E2P conformational state. Taken together, our results indicate that the phosphate bond of the phosphoenzyme intermediate of H(+)-ATPases is labile in the basal state, which may provide an explanation for the low H(+)/ATP coupling ratio of these pumps in the basal state.
authors.affiliation: From the Center for Membrane Pumps in Cells and Disease-PUMPKIN, Danish National Research Foundation, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark.
authors.forename: Jesper Torbøl
authors.initials: JT
authors.lastname: Pedersen
authors.affiliation: From the Center for Membrane Pumps in Cells and Disease-PUMPKIN, Danish National Research Foundation, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark.
authors.forename: Janus
authors.initials: J
authors.lastname: Falhof
authors.affiliation: From the Center for Membrane Pumps in Cells and Disease-PUMPKIN, Danish National Research Foundation, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark kiek@plen@ku.dk.
authors.forename: Kira
authors.initials: K
authors.lastname: Ekberg
authors.affiliation: From the Center for Membrane Pumps in Cells and Disease-PUMPKIN, Danish National Research Foundation, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark.
authors.forename: Morten Jeppe
authors.initials: MJ
authors.lastname: Buch-Pedersen
authors.affiliation: From the Center for Membrane Pumps in Cells and Disease-PUMPKIN, Danish National Research Foundation, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark palmgren@plen.ku.dk.
authors.forename: Michael
authors.initials: M
authors.lastname: Palmgren
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001608:Beryllium / Q000494:pharmacology*; D005459:Fluorides / Q000494:pharmacology*; D006868:Hydrolysis; D011499:Protein Processing, Post-Translational; D054328:Proton Pump Inhibitors / Q000494:pharmacology*; D017494:Proton Pumps / Q000187:drug effects* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000254:growth & development / Q000378:metabolism
chemical_list: D054328:Proton Pump Inhibitors; D017494:Proton Pumps; C035982:beryllium fluoride; D000255:Adenosine Triphosphate; D001608:Beryllium; D005459:Fluorides
keywords: H+-ATPase; plasma membrane; post-translational modification (PTM); proton pump; proton transport