DISTINCTIVE ROLES OF THE 2 ATP-BINDING SITES IN CLPA, THE ATPASE COMPONENT OF PROTEASE TI IN ESCHERICHIA-COLI

ClpA is the ATPase component of the ATP dependent protease Ti (Clp) in Escherichia coli and contains two ATP-binding sites, A ClpA variant ( referred to as ClpAT) carrying threonine in place of the 169th methion ine has recently been shown to be highly soluble but indistinguishable from the wild type, 84-kDa ClpA in its ability to hydrolyze ATP and t o support the casein-degrading activity of ClpP, Therefore, site-direc ted mutagenesis was performed to generate mutations in either of the t wo ATP-binding sites of ClpAT (i.e, to replace the Lys(220) or Lys(501 ) with Thr), ClpAT/K220T hydrolyzed ATP and supported the ClpP-mediate d proteolysis 10-50% as well as ClpAT depending on ATP concentration, while ClpAT/K501T was unable to cleave ATP or to support the proteolys is. Without ATP, ClpAT and both of its mutant forms behaved as trimeri c molecules as analyzed by gel filtration on a Sephacryl S-300 column, With 0.5 mM ATP, ClpAT and ClpAT/K501T became hexamers, but ClpAT/K22 0T remained trimeric, With 2 mM ATP, however, ClpAT/K220T also behaved as a hexamer, These results suggest that the first ATP-binding site o f ClpA is responsible for hexamer formation, while the second is essen tial for ATP hydrolysis, When trimeric ClpAT/K220T was incubated with the same amount of hexameric ClpAT/K501T (i,e, at 0.5 mM ATP) and then subjected to gel filtration as above, a majority of ClpAT/K220T ran t ogether with ClpAT/K501T as hexameric molecules. Furthermore, ClpAT/K5 01T in the mixture strongly inhibited the ability of ClpAT/K220T to cl eave ATP and to support the ClpP-mediated proteolysis, Similar results were obtained in the presence of 2 mM ATP and also with the mixture w ith ClpAT, On the other hand, the ATPase activity of the mixture of Cl pAT and ClpAT/R220T was significantly higher than the sum of that of e ach protein, particularly in the presence of 2 mM ATP, although its ab ility to support the proteolysis by ClpP remained un changed, These re sults suggest that a rapid exchange of the subunits, possibly as a tri meric unit, occurs between the ClpAT proteins in the presence of ATP a nd leads to the formation of mixed hexameric molecules.