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 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 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 similar to40 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.