ATP Hydrolysis and Synthesis of a Rotary Motor V-ATPase from Thermus thermophilus

Vacuolar-type H+-ATPase (V-ATPase) catalyzes ATP synthesis and hydrolysis coupled with proton translocation across membranes via a rotary motor mechanism. Here we report biochemical and biophysical catalytic properties of V-ATPase from Thermus thermophilus. ATP hydrolysis of V-ATPase was severely inhibited by entrapment of Mg-ADP in the catalytic site. In contrast, the enzyme was very active for ATP synthesis (∼70 s–1) with the Km values for ADP and phosphate being 4.7 ± 0.5 and 460 ± 30 μm, respectively. Single molecule observation showed V-ATPase rotated in a 120° stepwise manner, and analysis of dwelling time allowed the binding rate constant kon for ATP to be estimated (∼1.1 × 106m–1 s–1), which was much lower than the kon (= Vmax/Km) for ADP (∼1.4 × 107m–1 s–1). The slower konATP than konADP and strong Mg-ADP inhibition may contribute to prevent wasteful consumption of ATP under in vivo conditions when the proton motive force collapses.