J. Biochem, 1993, Vol. 114, No. 2 171-176
© 1993 Japanese Biochemical Society
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The 
Subunit of ATP Synthase (FoF1): 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 Interaction1
Department of Organic Chemistry and Biochemistry, The Institute of Scientific and Industrial Research, Osaka University Ibaraki, Osaka 567
The sequence (Gly-X-X-X-X-Gly-Lys-Thr/Ser) is conserved in nucleotide binding proteins including the 
and ß subunits of the ATP synthase. Various mutations were introduced in the Lys-175 and Thr-176 residues in the sequence (Gly-Asp-Arg-Gln-Thr-Gly-Lys-Thr, residues 169–176) of the Escherichia coli ATP synthase subunit. Surprisingly, single amino acid substitutions drastically affected the subunit assembly of the enzyme. The entire enzyme assembly was lost by Lys-175
Phe (or Trp) or Thr-176Phe (or Tyr) mutation. Other mutants had similar (His-175, Ser-175, Gly-175, Ser-176, and His-176 mutants) or lower (Ala-176, Cys-176, Leu-176, and Val-176 mutants) effects on assembly of the active enzyme compared with that of the wild-type. However, all these mutant enzymes except the Ser-176 enzyme showed enhanced cold sensitivities and reduced stabilities at high temperature. Mutant enzymes such as Gly-175 and His-176 showed low multi-site (steady state) catalysis, possibly due to loss of proper subunit-subunit interactions. These results suggest that the Lys-175 and Thr-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 subunit residues are different from those of the corresponding ß subunit residues (ßLys-155 and ßThr-156) which are essential for catalysis [Omote, H., Maeda, M., & Futai, M. (1992) J. Biol. Chem. 267, 20571–20576].
1 This study was supported by grants from the Ministry of Education, Science and Culture of Japan, and a grant from the Human Frontier Science Research Program Grant.
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