One rotary mechanism for F1-ATPase over ATP concentrations from millimolar down to nanomolar.
F(1)-ATPase is a rotary molecular motor in which the central gamma-subunit rotates inside a cylinder made of alpha(3)beta(3)-subunits. The rotation is driven by ATP hydrolysis in three catalytic sites on the beta-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 F(1) rotates at extremely low ATP concentrations where the site occupancy is expected to be low. We observed under an optical microscope rotation of individual F(1) 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 10(7) M(-1)s(-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. F(1) produced the same torque of approximately 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.
Sakaki
Naoyoshi
N
Department of Functional Molecular Science, The Graduate University for Advanced Studies, Nishigonaka 38, Myodaiji, Okazaki 444-8585, Japan.
Shimo-Kon
Rieko
R
Adachi
Kengo
K
Itoh
Hiroyasu
H
Furuike
Shou
S
Muneyuki
Eiro
E
Yoshida
Masasuke
M
Kinosita
Kazuhiko
K
D000255:Adenosine Triphosphate / Q000737:chemistry*; D006868:Hydrolysis; D007700:Kinetics; D008832:Microchemistry / Q000379:methods*; D020409:Molecular Motor Proteins / Q000737:chemistry*; D049329:Nanostructures / Q000737:chemistry* / Q000648:ultrastructure*; D011485:Protein Binding; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000648:ultrastructure*; D012399:Rotation; D019415:Torque
D020409:Molecular Motor Proteins; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases