ALANINE-SCANNING MUTAGENESIS OF THE EPSILON-SUBUNIT OF THE F-1-F-0 ATP SYNTHASE FROM ESCHERICHIA-COLI REVEALS 2 CLASSES OF MUTANTS

Alanine scanning mutagenesis was applied to the epsilon subunit of the F-1-F-0 ATP synthase from E. coli. Nineteen amino acid residues were changed to alanine, either singly or in pairs, between residues 10 and 93. All mutants, when expressed in the epsilon deletion strain XH1, w ere able to grow on succinate minimal medium. Membranes were prepared from all mutants and assayed for ATP-driven proton translocation, ATP hydrolysis +/- lauryldiethylamine oxide, and sensitivity of ATPase act ivity to N,N'-dicyclohexylcarbodiimide (DCCD). Most of the mutants fel l into 2 distinct classes. The first group had inhibited ATPase activi ty, with near normal levels of membrane bound F-1, but decreased sensi tivity to DCCD. The second group had stimulated ATPase activity, with a reduced level of membrane-bound F-1, but normal sensitivity to DCCD. Membranes from all mutants were further characterized by immunoblotti ng using 2 monoclonal antibodies. A model for the secondary structure of epsilon and its role in the function of the ATP synthase has been d eveloped. Some residues are important for the binding of epsilon to F- 1 and therefore for inhibition. Other residues, from Glu-59 through Gl u-70, are important for the release of inhibition by epsilon that is p art of the normal enzyme cycle.