title: Rotational catalysis of Escherichia coli ATP synthase F1 sector. Stochastic fluctuation and a key domain of the beta subunit.
abstract: A complex of gamma, epsilon, and c subunits rotates in ATP synthase (FoF(1)) coupled with proton transport. A gold bead connected to the gamma subunit of the Escherichia coli F(1) sector exhibited stochastic rotation, confirming a previous study (Nakanishi-Matsui, M., Kashiwagi, S., Hosokawa, H., Cipriano, D. J., Dunn, S. D., Wada, Y., and Futai, M. (2006) J. Biol. Chem. 281, 4126-4131). A similar approach was taken for mutations in the beta subunit key region; consistent with its bulk phase ATPase activities, F(1) with the Ser-174 to Phe substitution (betaS174F) exhibited a slower single revolution time (time required for 360 degree revolution) and paused almost 10 times longer than the wild type at one of the three 120 degrees positions during the stepped revolution. The pause positions were probably not at the "ATP waiting" dwell but at the "ATP hydrolysis/product release" dwell, since the ATP concentration used for the assay was approximately 30-fold higher than the K(m) value for ATP. A betaGly-149 to Ala substitution in the phosphate binding P-loop suppressed the defect of betaS174F. The revertant (betaG149A/betaS174F) exhibited similar rotation to the wild type, except that it showed long pauses less frequently. Essentially the same results were obtained with the Ser-174 to Leu substitution and the corresponding revertant betaG149A/betaS174L. These results indicate that the domain between beta-sheet 4 (betaSer-174) and P-loop (betaGly-149) is important to drive rotation.
authors.affiliation: Futai Special Laboratory, Microbial Chemistry Research Center, Microbial Chemistry Research Foundation, Tokyo 141-0021, Japan.
authors.forename: Mayumi
authors.initials: M
authors.lastname: Nakanishi-Matsui
authors.forename: Sachiko
authors.initials: S
authors.lastname: Kashiwagi
authors.forename: Toshiharu
authors.initials: T
authors.lastname: Ubukata
authors.forename: Atsuko
authors.initials: A
authors.lastname: Iwamoto-Kihara
authors.forename: Yoh
authors.initials: Y
authors.lastname: Wada
authors.forename: Masamitsu
authors.initials: M
authors.lastname: Futai
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D019943:Amino Acid Substitution; D025243:Bacterial Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D008958:Models, Molecular; D020125:Mutation, Missense; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; D025243:Bacterial Proton-Translocating ATPases