The alpha/beta subunit interaction in H(+)-ATPase (ATP synthase). An Escherichia coli alpha subunit mutation (Arg-alpha 296-->Cys) restores coupling efficiency to the deleterious beta subunit mutant (Ser-beta 174-->Phe).
The Ser-beta 174 residue of the Escherichia coli H(+)-ATPase beta subunit has been shown to be near the catalytic site together with Gly-beta 149, Gly-beta 172, Glu-beta 192, and Val-beta 198 (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-beta 174 mutant was defective in energy coupling between catalysis and transport, whereas the Leu-beta 174 mutant could couple efficiently, although both mutants had essentially the same ATPase activities (approximately 10% of the wild type). The defective energy coupling of the Phe-beta 174 mutant was suppressed by the second mutation (Arg-alpha 296-->Cys) in the alpha subunit. The Cys-alpha 296/Phe-beta 174 mutant had essentially the same membrane ATPase activity as the Phe-beta 174 single mutant when assayed under the conditions that stabilize the double mutant enzyme. These results indicate the importance of the alpha/beta interaction, especially that between the regions near Arg-alpha 296 and Ser-beta 174, for energy coupling in the H(+)-ATPase. The 2 residues (Ser-beta 174 and Arg-alpha 296) 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 alpha subunit of Cys-alpha 296/Phe-beta 174 or Cys-alpha 296 mutant ATPase, but could not inhibit the enzyme activity. This is the first intersubunit mutation/suppression approach to ATPase catalysis and its energy coupling.
Omote
H
H
Department of Organic Chemistry and Biochemistry, Osaka University, Japan.
Park
M Y
MY
Maeda
M
M
Futai
M
M
D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics; D001692:Biological Transport; D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology; D003545:Cysteine / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D006859:Hydrogen / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010649:Phenylalanine / Q000235:genetics; D006180:Proton-Translocating ATPases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics
D012694:Serine; D010649:Phenylalanine; D006859:Hydrogen; D001120:Arginine; D006180:Proton-Translocating ATPases; D003545:Cysteine; D005033:Ethylmaleimide