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Single-molecule Study on the Temperature-sensitive Reaction of F(1)-ATPase with a Hybrid F(1) Carrying a Single β(E190D)

F(1)-ATPase is a rotary molecular motor in which the γ-subunit rotates against the α(3)β(3) cylinder. The unitary γ-rotation is a 120° step comprising 80 and 40° substeps, each of these initiated by ATP binding and ADP release and by ATP hydrolysis and inorganic phosphate release, respectively. In o...

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Autores principales: Enoki, Sawako, Watanabe, Rikiya, Iino, Ryota, Noji, Hiroyuki
Formato: Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2755722/
https://www.ncbi.nlm.nih.gov/pubmed/19561076
http://dx.doi.org/10.1074/jbc.M109.026401
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author Enoki, Sawako
Watanabe, Rikiya
Iino, Ryota
Noji, Hiroyuki
author_facet Enoki, Sawako
Watanabe, Rikiya
Iino, Ryota
Noji, Hiroyuki
author_sort Enoki, Sawako
collection PubMed
description F(1)-ATPase is a rotary molecular motor in which the γ-subunit rotates against the α(3)β(3) cylinder. The unitary γ-rotation is a 120° step comprising 80 and 40° substeps, each of these initiated by ATP binding and ADP release and by ATP hydrolysis and inorganic phosphate release, respectively. In our previous study on γ-rotation at low temperatures, a highly temperature-sensitive (TS) reaction step of F(1)-ATPase from thermophilic Bacillus PS3 was found below 9 °C as an intervening pause before the 80° substep at the same angle for ATP binding and ADP release. However, it remains unclear as to which reaction step the TS reaction corresponds. In this study, we found that the mutant F(1)(βE190D) from thermophilic Bacillus PS3 showed a clear pause of the TS reaction below 18 °C. In an attempt to identify the catalytic state of the TS reaction, the rotation of the hybrid F(1), carrying a single copy of βE190D, was observed at 18 °C. The hybrid F(1) showed a pause of the TS reaction at the same angle as for the ATP binding of the incorporated βE190D, although kinetic analysis revealed that the TS reaction is not the ATP binding step. These findings suggest that the TS reaction is a structural rearrangement of β before or after ATP binding.
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spelling pubmed-27557222009-10-13 Single-molecule Study on the Temperature-sensitive Reaction of F(1)-ATPase with a Hybrid F(1) Carrying a Single β(E190D) Enoki, Sawako Watanabe, Rikiya Iino, Ryota Noji, Hiroyuki J Biol Chem Metabolism and Bioenergetics F(1)-ATPase is a rotary molecular motor in which the γ-subunit rotates against the α(3)β(3) cylinder. The unitary γ-rotation is a 120° step comprising 80 and 40° substeps, each of these initiated by ATP binding and ADP release and by ATP hydrolysis and inorganic phosphate release, respectively. In our previous study on γ-rotation at low temperatures, a highly temperature-sensitive (TS) reaction step of F(1)-ATPase from thermophilic Bacillus PS3 was found below 9 °C as an intervening pause before the 80° substep at the same angle for ATP binding and ADP release. However, it remains unclear as to which reaction step the TS reaction corresponds. In this study, we found that the mutant F(1)(βE190D) from thermophilic Bacillus PS3 showed a clear pause of the TS reaction below 18 °C. In an attempt to identify the catalytic state of the TS reaction, the rotation of the hybrid F(1), carrying a single copy of βE190D, was observed at 18 °C. The hybrid F(1) showed a pause of the TS reaction at the same angle as for the ATP binding of the incorporated βE190D, although kinetic analysis revealed that the TS reaction is not the ATP binding step. These findings suggest that the TS reaction is a structural rearrangement of β before or after ATP binding. American Society for Biochemistry and Molecular Biology 2009-08-21 2009-06-26 /pmc/articles/PMC2755722/ /pubmed/19561076 http://dx.doi.org/10.1074/jbc.M109.026401 Text en © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Author's Choice—Final version full access. Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) applies to Author Choice Articles
spellingShingle Metabolism and Bioenergetics
Enoki, Sawako
Watanabe, Rikiya
Iino, Ryota
Noji, Hiroyuki
Single-molecule Study on the Temperature-sensitive Reaction of F(1)-ATPase with a Hybrid F(1) Carrying a Single β(E190D)
title Single-molecule Study on the Temperature-sensitive Reaction of F(1)-ATPase with a Hybrid F(1) Carrying a Single β(E190D)
title_full Single-molecule Study on the Temperature-sensitive Reaction of F(1)-ATPase with a Hybrid F(1) Carrying a Single β(E190D)
title_fullStr Single-molecule Study on the Temperature-sensitive Reaction of F(1)-ATPase with a Hybrid F(1) Carrying a Single β(E190D)
title_full_unstemmed Single-molecule Study on the Temperature-sensitive Reaction of F(1)-ATPase with a Hybrid F(1) Carrying a Single β(E190D)
title_short Single-molecule Study on the Temperature-sensitive Reaction of F(1)-ATPase with a Hybrid F(1) Carrying a Single β(E190D)
title_sort single-molecule study on the temperature-sensitive reaction of f(1)-atpase with a hybrid f(1) carrying a single β(e190d)
topic Metabolism and Bioenergetics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2755722/
https://www.ncbi.nlm.nih.gov/pubmed/19561076
http://dx.doi.org/10.1074/jbc.M109.026401
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