Allosteric effects of RuvA protein, ATP, and DNA on RuvB protein-mediated ATP hydrolysis

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 k(cat) and decreases the K-m 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 [Marrione & Cox(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 K-m. 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.