Role of the ε subunit of thermophilic F1-ATPase as a sensor for ATP

Abstract

The ε subunit of F1-ATPase from the thermophilic Bacillus PS3 (TF1) has been shown to bind ATP. The precise nature of the regulatory role of ATP binding to the ε subunit remains to be determined. To address this question, 11 mutants of the ε subunit were prepared, in which one of the basic or acidic residues was substituted with alanine. ATP binding to these mutants was tested by gel-filtration chromatography. Among them, four mutants that showed no ATP binding were selected and reconstituted with the α3β3γ complex of TF1. The ATPase activity of the resulting α3β3γε complexes was measured, and the extent of inhibition by the mutant εsubunits was compared in each case. With one exception, weaker binding of ATP correlated with greater inhibition of ATPase activity. These results clearly indicate that ATP binding to the ε subunit plays a regulatory role and that ATP binding may stabilize the ATPase-active form of TF1 by fixing the ε subunit into the folded conformation.