DnaK, the prototype Hsp70 protein of Escherichia coli, functions as a molecular chaperone in protein folding disassembly reactions through cycles of polypeptides binding and release that are coupled to its intrinsic ATPase activity. To further our understanding of these processes, we sought to obtain a quantitative description of the basic ATPase cycle of DnaK. To this end, we have performed steady-state and pre-steady-state kinetics experiments and have determined rate constants corresponding to individual steps in the DnaK ATPase cycle at 25 °C. Hydrolysis of ATP proceeds very slowly with a rate constant (k(hyd) ≃ 0.02 min-1) at least 10-fold smaller than the rate constant for any other first-order step in the forward reaction pathway. The ATP hydrolysis step has an activation energy of 26.2 ± 0.4 kcal/mol and is rate limiting in the steady-state under typical in vitro conditions. ATP binds with unusual strength to DnaK, with a measured K(D) ≃ 1 nM. ADP binds considerably less tightly than ATP). However, in the presence of physiologically relevant concentrations of inorganic phosphate (P(i)), the release of ADP from DnaK is greatly slowed, approximately to the rate of ATP hydrolysis. Under these conditions, the ADP-bound form of DnaK, the form that binds substrate polypeptidase most tightly, was found to represent a significant fraction of the DnaK population. The slowing of ADP release by exogenous P(i) is due to thermodynamic coupling of the binding of the two ligands, which produces a coupling energy of ~1.6 kcal/mol. This result implies that product release is not strictly ordered. In the absence of exogenous inorganic phosphate, P(i) product, by virtue of its higher k(off), is released prior to ADP. However, at physiological concentrations of inorganic phosphate, the alternate product release pathway, whereby ADP dissociates from a ternary DnaK·ADP·P(i) complex, become more prominent.
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