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Originally published as Biophys J. BioFAST on December 30, 2004.
doi:10.1529/biophysj.104.054668
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Biophysical Journal 88:2047-2056 (2005)
© 2005 The Biophysical Society

One Rotary Mechanism for F1-ATPase over ATP Concentrations from Millimolar down to Nanomolar

Naoyoshi Sakaki * {dagger}, Rieko Shimo-Kon {dagger}, Kengo Adachi {dagger}, Hiroyasu Itoh {ddagger} §, Shou Furuike {dagger}, Eiro Muneyuki ¶, Masasuke Yoshida ¶ || and Kazuhiko Kinosita, Jr. {dagger}

* Department of Functional Molecular Science, The Graduate University for Advanced Studies, Nishigonaka 38, Myodaiji, Okazaki 444-8585, Japan; {dagger} Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Higashiyama 5-1, Myodaiji, Okazaki 444-8787, Japan; {ddagger} Tsukuba Research Laboratory, Hamamatsu Photonics KK, and § CREST "Creation and Application of Soft Nano-Machine, the Hyperfunctional Molecular Machine" Team 13*, Tokodai, Tsukuba 300-2635, Japan; Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259, Yokohama 226-8503, Japan; and || ERATO "ATP System", Japan Science and Technology Agency, Nagatsuta 5800-3, Yokohama 226-0026, Japan

Correspondence: Address reprint requests to Kazuhiko Kinosita Jr., Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Higashiyama 5-1, Myodaiji, Okazaki 444-8787, Japan. Tel.: 81-564-59-5230; Fax: 81-564-59-5234; E-mail: kazuhiko{at}ims.ac.jp.

F1-ATPase is a rotary molecular motor in which the central {gamma}-subunit rotates inside a cylinder made of {alpha}3ß3-subunits. The rotation is driven by ATP hydrolysis in three catalytic sites on the ß-subunits. How many of the three catalytic sites are filled with a nucleotide during the course of rotation is an important yet unsettled question. Here we inquire whether F1 rotates at extremely low ATP concentrations where the site occupancy is expected to be low. We observed under an optical microscope rotation of individual F1 molecules that carried a bead duplex on the {gamma}-subunit. Time-averaged rotation rate was proportional to the ATP concentration down to 200 pM, giving an apparent rate constant for ATP binding of 2 x 107 M–1s–1. A similar rate constant characterized bulk ATP hydrolysis in solution, which obeyed a simple Michaelis-Menten scheme between 6 mM and 60 nM ATP. F1 produced the same torque of ~40 pN·nm at 2 mM, 60 nM, and 2 nM ATP. These results point to one rotary mechanism governing the entire range of nanomolar to millimolar ATP, although a switchover between two mechanisms cannot be dismissed. Below 1 nM ATP, we observed less regular rotations, indicative of the appearance of another reaction scheme.




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