THE ALPHA-SUBUNIT OF ATP SYNTHASE (F0F1) - THE LYS-175 AND THR-176 RESIDUES IN THE CONSERVED SEQUENCE (GLY-X-X-X-X-GLY-LYS-THR-SER) ARE LOCATED IN THE DOMAIN REQUIRED FOR STABLE SUBUNIT-SUBUNIT INTERACTION

The sequence (Gly-X-X-X-X-Gly-Lys-Thr/Ser) is conserved in nucleotide binding proteins including the alpha and beta subunits of the ATP synthase. Various mutations were introduced in the alphaLys-175 and alphaThr-176 residues in the sequence (Gly-Asp-Arg-Gln-Thr-Gly-Lys-Thr, residues 169-176) of the Escherichia coli ATP synthase alpha subunit. Surprisingly, single amino acid substitutions drastically affected the subunit assembly of the enzyme. The entire enzyme assembly was lost by alphaLys-175-->Phe (or Trp) or alphaThr-176-->Phe (or Tyr) mutation. Other mutants had similar (alphaHis-175, alphaSer-175, alphaGly-175, alphaSer-176, and alphaHis-176 mutants) or lower (alphaAla-176, alphaCys-176, alphaLeu-176, and alphaVal-176 mutants) effects on assembly of the active enzyme compared with that of the wild-type. However, all these mutant enzymes except the alphaSer-176 enzyme showed enhanced cold sensitivities and reduced stabilities at high temperature. Mutant enzymes such as alphaGly-175 and alphaHis-176 showed low multi-site (steady state) catalysis, possibly due to loss of proper subunit-subunit interactions. These results suggest that the alphaLys-175 and alphaThr-176 residues are not absolutely essential for catalysis, but that they, or possibly the entire conserved sequence, are located in the key domain for the subunit-subunit interactions essential for enzyme stability and steady state activity. The roles of the alpha subunit residues are different from those of the corresponding beta subunit residues (betaLys-155 and betaThr-156) which are essential for catalysis [Omote, H., Maeda, M., & Futai, M. (1992) J. Biol. Chem. 267, 20571-20576].