Mutations in Ser174 and the glycine-rich sequence (Gly149, Gly150, and Thr156) in the beta subunit of Escherichia coli H(+)-ATPase.
A sequence motif in the beta subunit of Escherichia coli F1 (Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr, residue 149-156, where conserved residues are underlined) is one of the glycine-rich sequences found in many nucleotide binding proteins. In this study, we constructed a plasmid carrying all the F0F1 genes. This plasmid gave the highest membrane ATPase activity so far reported. Substitution of beta Gly149 by Ser suppressed the effect of the beta Ser174----Phe mutation (defective H(+)-ATPase), but beta Gly150----Ser substitution did not have this effect. A single mutation (beta Gly149----Ser or beta Gly150----Ser) gave active enzyme with altered divalent cation dependency and azide sensitivity: the beta Gly149----Ser mutant enzyme had 100-fold lower azide sensitivity and essentially no Ca(2+)-dependent activity, but had the wild-type level of Mg(2+)-dependent activity with active oxidative phosphorylation. Introduction of a beta Gly149----Ser or beta Gly150----Ser mutation with the beta Ser174----Phe mutation also lowered the Ca(2+)-dependent activity and azide sensitivity. Consistent with our previous findings (Takeyama, M., Ihara, K., Moriyama, Y., Noumi, T., Ida, K., Tomioka, N., Itai, A., Maeda, M., and Futai, M. (1990) J. Biol. Chem. 265, 21279-21284), a beta Thr156----Ala or Cys mutation impaired ATPase activity, suggesting that the hydroxyl moiety at position 156 is essential for the catalytic activity. The possible location of the catalytic site including divalent cation binding site(s) is discussed.
Iwamoto
A
A
Institute of Scientific and Industrial Research, Osaka University, Japan.
Omote
H
H
Hanada
H
H
Tomioka
N
N
Itai
A
A
Maeda
M
M
Futai
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D017301:Ca(2+) Mg(2+)-ATPase / Q000235:genetics; D000252:Calcium-Transporting ATPases / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005998:Glycine / Q000737:chemistry*; D008969:Molecular Sequence Data; D009154:Mutation; D009876:Operon; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics*; D016415:Sequence Alignment; D012694:Serine / Q000737:chemistry*
D004269:DNA, Bacterial; D012694:Serine; D017301:Ca(2+) Mg(2+)-ATPase; D006180:Proton-Translocating ATPases; D000252:Calcium-Transporting ATPases; D005998:Glycine