Escherichia coli F0F1-ATPase. Residues involved in catalysis and coupling.

R. K. Nakamoto, K. Shin, A. Iwamoto, Hiroshi Omote, M. Maeda, M. Futai

Research output: Contribution to journalArticle

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Abstract

The molecular biological approach has provided important information toward understanding the complexities of the F0F1 ATPase. This article focuses on our recent results on the ATPase catalytic site contained in the beta subunit and the role of the gamma subunit in regulation of proton transport. We used a combination of affinity labeling and mutagenesis to locate several residues of the alpha and beta subunits in the catalytic site. Adenosine triphosphopyridoxal (AP3-PL) labeled beta Lys-155, beta Lys-201 and alpha Lys-201, suggesting that they are near the gamma-phosphate moiety of ATP. Turning to a mutagenesis approach we demonstrated that the two conserved residues, beta Lys-155 and beta Thr-156 in the glycine-rich sequence, are essential for catalysis. Finally, using pseudorevertant analysis, we positioned residue beta Gly-149 (also in the glycine-rich sequence) in proximity to beta Ser-174, beta Glu-192 (binding site for DCCD), and beta Val-198 (only three residues away from the AP3-PL binding site, beta Lys-201). Genetic studies suggested that the gamma subunit plays a role in regulation of catalysis and its coupling with proton conduction. We found that four mutations in the carboxyl-terminal region (gamma Gln-269-->Leu, gamma Gly-275-->Lys, gamma Thr-277-->end, or frameshift) had similar membrane ATPase activities but different ATP-dependent proton pumping and growth by oxidative phosphorylation. These results suggested a perturbation in the coupling between catalysis and proton translocation. We were able to clearly define the "uncoupling" by introducing mutations in the amino-terminal region of the gamma subunit. We were led to gamma Met-23-->Lys and Arg which resulted in an enzyme still regulated by delta microH+, but with profoundly inefficient coupling between ATPase catalytic sites and proton translocation in both ATP-dependent proton pumping and delta microH(+)-driven ATP synthesis. Second-site mutations in the carboxyl-terminal region of the gamma subunit reversed this effect.

Original languageEnglish
Pages (from-to)335-343
Number of pages9
JournalAnnals of the New York Academy of Sciences
Volume671
Publication statusPublished - Nov 30 1992
Externally publishedYes

Fingerprint

Proton-Translocating ATPases
Catalysis
Escherichia coli
Protons
Adenosine Triphosphate
Adenosine Triphosphatases
Catalytic Domain
Mutagenesis
Glycine
Mutation
Binding Sites
Dicyclohexylcarbodiimide
Oxidative Phosphorylation
Labeling
Phosphates
Membranes
Enzymes
Growth
adenosine triphosphopyridoxal

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Nakamoto, R. K., Shin, K., Iwamoto, A., Omote, H., Maeda, M., & Futai, M. (1992). Escherichia coli F0F1-ATPase. Residues involved in catalysis and coupling. Annals of the New York Academy of Sciences, 671, 335-343.

Escherichia coli F0F1-ATPase. Residues involved in catalysis and coupling. / Nakamoto, R. K.; Shin, K.; Iwamoto, A.; Omote, Hiroshi; Maeda, M.; Futai, M.

In: Annals of the New York Academy of Sciences, Vol. 671, 30.11.1992, p. 335-343.

Research output: Contribution to journalArticle

Nakamoto, RK, Shin, K, Iwamoto, A, Omote, H, Maeda, M & Futai, M 1992, 'Escherichia coli F0F1-ATPase. Residues involved in catalysis and coupling.', Annals of the New York Academy of Sciences, vol. 671, pp. 335-343.
Nakamoto, R. K. ; Shin, K. ; Iwamoto, A. ; Omote, Hiroshi ; Maeda, M. ; Futai, M. / Escherichia coli F0F1-ATPase. Residues involved in catalysis and coupling. In: Annals of the New York Academy of Sciences. 1992 ; Vol. 671. pp. 335-343.
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