GRPE ALTERS THE AFFINITY OF DNAK FOR ATP AND MG2- IMPLICATIONS FOR THE MECHANISM OF NUCLEOTIDE EXCHANGE( )

DnaK, DnaJ, and GrpE heat shock proteins of Escherichia coli activate site-specific DNA binding by the RepA replication initiator protein of plasmid P1 in a reaction dependent on ATP and Mg2+. We previously sho wed that GrpE is essential for in. vitro RepA activation specifically at about 1 mu m free Mg2+. In this paper, we demonstrate that GrpE low ers the requirement of DnaK ATPase for Mg2+, resulting in a large stim ulation of ATP hydrolysis at about 1 mu M Mg2+ with and without DnaJ a nd RepA. In contrast to its effect on the Mg2+ requirement, GrpE incre ases the ATP requirement for DnaK ATPase and dramatically lowers the a ffinity of DnaK for ATP in the absence of Mg2+. We propose that GrpE n ot only lowers the affinity of DnaK for nucleotide but, by increasing affinity of DnaK for Mg2+, also weakens the interactions of Mg2+ with nucleotide prior to its release.