PMID:9712846
| Citation |
Ketchum, CJ and Nakamoto, RK (1998) A mutation in the Escherichia coli F0F1-ATP synthase rotor, gammaE208K, perturbs conformational coupling between transport and catalysis. J. Biol. Chem. 273:22292-7 |
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| Abstract |
Cross-linking studies on the Escherichia coli F0F1-ATP synthase indicated a site of interaction involving gamma and epsilon subunits in F1 and subunit c in F0 (Watts, S. D., Tang, C., and Capaldi, R. A. (1996) J. Biol. Chem. 271, 28341-28347). To assess the function of these interactions, we introduced random mutations in this region of the gamma subunit (gamma194-213). One mutation, gammaGlu-208 to Lys (gammaE208K), caused a temperature-sensitive defect in oxidative phosphorylation-dependent growth. ATP hydrolytic rates of the gammaE208K F0F1 enzyme became increasingly uncoupled from H+ pumping above 28 degreesC. In contrast, Arrhenius plot of steady-state ATP hydrolysis of the mutant enzyme was linear from 20 to 50 degreesC. Analysis of this plot revealed a significant increase in the activation energy of the catalytic transition state to a value very similar to soluble, epsilon subunit-inhibited F1 and suggested that the mutation blocked normal release of epsilon inhibition of ATP hydrolytic activity upon binding of F1 to F0. The difference in temperature dependence suggested that the gammaE208K mutation perturbed release of inhibition via a different mechanism than it did energy coupling. Suppressor mutations in the polar loop of subunit c restored ATP-dependent H+ pumping and transition state thermodynamic parameters close to wild-type values indicating that interactions between gamma and c subunits mediate release of epsilon inhibition and communication of coupling information. |
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| Keywords |
Catalysis; Escherichia coli/enzymology; Escherichia coli/growth & development; Ion Transport; Mutagenesis, Site-Directed; Oxidative Phosphorylation; Protein Binding; Protein Conformation; Proton-Translocating ATPases/antagonists & inhibitors; Proton-Translocating ATPases/genetics; Proton-Translocating ATPases/metabolism; Protons; Temperature |
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