The Ser-β174 residue of the Escherichia coli H+-ATPase β subunit has been shown to be near the catalytic site together with Gly-β149, Gly-β172, Glu-β192, and Val-β198 (Iwamoto, A., Park, M.-Y., Maeda, M., and Futai, M. (1993) J. Biol. Chem. 268, 3156-3160). In this study, we introduced various residues at position 174 and found that the larger the side chain volume of the residue introduced, the lower the enzyme activity became. The Phe-β174 mutant was defective in energy coupling between catalysis and transport, whereas the Leu-β174 mutant could couple efficiently, although both mutants had essentially the same ATPase activities (~10% of the wild type). The defective energy coupling of the Phe-β174 mutant was suppressed by the second mutation (Arg-α296 → Cys) in the α subunit. The Cys-α296/Phe- β174 mutant had essentially the same membrane ATPase activity as the Phe- β174 single mutant when assayed under the conditions that stabilize the double mutant enzyme. These results indicate the importance of the α/β interaction, especially that between the regions near Arg-α296 and Ser- β174, for energy coupling in the H+-ATPase. The 2 residues (Ser-β174 and Arg-α296) may be located nearby at the interface of the two subunits. About 1 mol of N-[14C]ethylmaleimide could bind to 1 mol of the α subunit of Cys-α296/Phe-β174 or Cys-α296 mutant ATPase, but could not inhibit the enzyme activity. This is the first intersubunit mutation/suppression approach to ATPase catalysis and its energy coupling.
|Number of pages||5|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - Jan 1 1994|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology