title: RuvB protein-mediated ATP hydrolysis: functional asymmetry in the RuvB hexamer.
abstract: A survey of RuvB protein-mediated ATP hydrolysis yields the following observations. (1) The RuvB protein exhibits a DNA-independent ATPase activity with a turnover number (based on a RuvB monomer) approaching 6 min-1 and a Km of 154 microM. Single-stranded DNA and linear duplex DNA have small but significant effects on this activity. (2) At ATP concentrations near the Km, the ATPase activity is attenuated after approximately 60 turnovers/RuvB monomer. The attenuation does not reflect inhibition by ADP. Addition of ATP to 3 mM triggers an immediate resumption of ATP hydrolysis. The attention is enhanced somewhat by ssDNA and reduced somewhat by linear dsDNA. (3) ATP hydrolysis is dramatically stimulated by circular dsDNA, reinforcing the notion that RuvB translocates along the DNA in a reaction coupled to ATP hydrolysis. The kcat increases by at least 2-4-fold on circular duplexes depending on conditions, and the inactivation of RuvB at ATP concentrations near the Km does not occur. The ATPase activity on circular dsDNA also exhibits a partial substrate inhibition by ATP. (4) Optimal ATP hydrolysis requires approximately 1 DNA circle/RuvB hexamer, suggesting that multiple RuvB hexamers on a circle have an inhibitory effect on the ATPase activity. (5) With or without any of these DNA cofactors, a burst of ATP hydrolysis is observed under pre-steady-state conditions equivalent to 1 ATP per 3-3.3 RuvB monomers (2 ATP/hexamer). The substrate inhibition and burst results suggest the presence of nonequivalent ATP hydrolytic sites in a RuvB hexamer. The attenuation of ATPase activity observed under some conditions may also be a manifestation of nonequivalent ATP hydrolytic sites.
authors.affiliation: Department of Biochemistry, College of Agriculture and Life Sciences, University of Wisconsin, Madison 53706, USA.
authors.forename: P E
authors.initials: PE
authors.lastname: Marrione
authors.forename: M M
authors.initials: MM
authors.lastname: Cox
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D004247:DNA / Q000378:metabolism / Q000494:pharmacology; D004260:DNA Repair; D004270:DNA, Circular / Q000378:metabolism / Q000494:pharmacology; D004277:DNA, Single-Stranded / Q000378:metabolism / Q000494:pharmacology; D006868:Hydrolysis; D007700:Kinetics; D046911:Macromolecular Substances; D013053:Spectrophotometry
chemical_list: D001426:Bacterial Proteins; D004270:DNA, Circular; D004277:DNA, Single-Stranded; D046911:Macromolecular Substances; C061260:RuvB protein, Bacteria; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases