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

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

^* Department of Functional Molecular Science, The Graduate University for Advanced Studies, Nishigonaka 38, Myodaiji, Okazaki 444-8585, Japan; Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Higashiyama 5-1, Myodaiji, Okazaki 444-8787, Japan; 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@ims.ac.jp.

F₁-ATPase is a rotary molecular motor in which the central -subunit rotates inside a cylinder made of 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 -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.