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 alpha Lys-175 and alpha Thr-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 alpha Lys-175-->Phe (or Trp) or alpha Thr-176-->Phe (or Tyr) mutation. Other mutants had similar (alpha His-175, alpha Ser-175, alpha Gly-175, alpha Ser-176, and alpha His-176 mutants) or lower (alpha Ala-176, alpha Cys-176, alpha Leu-176, and alpha Val-176 mutants) effects on assembly of the active enzyme compared with that of the wild-type. However, all these mutant enzymes except the alpha Ser-176 enzyme showed enhanced cold sensitivities and reduced stabilities at high temperature. Mutant enzymes such as alpha Gly-175 and alpha His-176 showed low multi-site (steady state) catalysis, possibly due to loss of proper subunit-subunit interactions. These results suggest that the alpha Lys-175 and alpha 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Jounouchi
M
M
Department of Organic Chemistry and Biochemistry, Osaka University.
Maeda
M
M
Futai
M
M
D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001483:Base Sequence; D003080:Cold Temperature; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D016133:Polymerase Chain Reaction; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases