HYDROLYSIS OF ADENOSINE 5'-TRIPHOSPHATE BY ESCHERICHIA-COLI GROEL - EFFECTS OF GROES AND POTASSIUM-ION

The potassium-ion activation constant (K(act)) for the ATPase activity of Escherichia coli chaperonin groEL is inversely dependent upon the ATP concentration over at least 3 orders of magnitude. The ATPase acti vity shows positively cooperative kinetics with respect to ATP and K+. Both the K0.5 for ATP and cooperativity (as measured by the Hill coef ficient) decrease as the K+ concentration increases. Equilibrium bindi ng studies under conditions where hydrolysis does not occur indicate t hat MgATP binds weakly to groEL in the absence of K+. In the absence o f groES, the K+-dependent hydrolysis of ATP by groEL continues to comp letion. In the presence of groES, the time course for the hydrolysis o f ATP by groEL becomes more complex. Three distinct kinetic phases can be discerned. Initially, both heptameric toroids turn over once at th e same rate that they do in the absence of groES. This leads to the fo rmation of an asymmetric binary complex, groEL14-MgADP7-groES7, in whi ch 7 mol of ADP is trapped in a form that does not readily exchange wi th free ADP. In the second phase, the remaining seven sites (containin g readily exchangeable ADP) turn over, or have the potential to turn o ver, at the same rate as they do in the absence of groES, so that the overall rate of hydrolysis is maximally 50%. These remaining sites of the asymmetric binary complex do not hydrolyze all of the available AT P. Instead, the second phase of hydrolysis gives way to a third, compl etely inhibited state, the onset of which is dependent upon the relati ve affinities of the remaining sites for MgATP and MgADP. This fully i nhibited state may be returned to the 50% inhibited state by increasin g the ATP/ADP ratio or by increasing the relative affinity of the avai lable sites for MgATP relative to MgADP (by increasing the K+ concentr ation). At low K+ concentrations (1 mM) it is possible to create the c ompletely inhibited state in the absence of free MgADP; the apparent a ffinity of this asymmetric binary complex for MgATP is reduced 1 order of magnitude.