A detailed characterization of RuvB protein-mediated ATP hydrolysis in the presence of RuvA protein has provided (a) the steady-state kinetic parameters of ATP hydrolysis within a RuvAB complex and (b) several insights into the mechanism of ATP hydrolysis and its coupling to translocation on DNA. In general, the RuvA protein increases the kcat and decreases the Km for the RuvB ATPase activity. DNA has a much greater effect on the kinetics of ATP hydrolysis when RuvA is present, consistent with a role of RuvA in facilitating the interaction between RuvB and DNA. Mechanistic clues come from deviations from normal steady-state kinetic behavior. A previously described burst of ATP hydrolysis, corresponding to two ATPs per RuvB hexamer [MarrioneCox (1995) Biochemistry 34, 9809-9818], is still observed in the presence of RuvA protein. This suggests a functional asymmetry in the RuvB hexamer. There is a gradual attenuation of ATP hydrolysis when RuvB protein, alone or in the presence of RuvA protein, hydrolyzes ATP at ATP concentrations below the Km. The attenuation is observed even though an ATP regeneration system is present. ATP hydrolysis simply halts after a limited number of turnovers. The attenuation is reversible, and the effects of RuvA protein, DNA, and additional ATP in reversing the effect provide evidence for a complex array of allosteric interactions operating within the RuvB hexameric helicase. We propose a model in which individual subunits in a RuvB hexamer are functionally paired, with the three pairs moving sequentially and cooperatively through a multistep ATP hydrolytic cycle.
