Kinetics of the Reactions of the Escherichia coli Molecular Chaperone DnaK with ATP:  Evidence That a Three-Step Reaction Precedes ATP Hydrolysis

The mechanism of the ATPase cycle of the 70-kDa Escherichia coli molecular chaperone DnaK was investigated by following ATP-induced changes in the tryptophan fluorescence of DnaK. Three steps in the cycle were investigated. (i) Stopped-flow experiments revealed that ATP induces a biphasic reduction in the tryptophan fluorescence of DnaK. The rate of the fast fluorescence transition exhibited a hyperbolic dependence on the ATP concentration, with a maximum rate equal to 56 (± 10) s-1 at 35 °C, whereas the rate of the slow fluorescence transition was nearly independent of the ATP concentration (4.2 ± 0.2 s-1). These results are consistent with the three-step sequential reaction E + ATP ⇔ E−ATP ⇔ E*−ATP ⇔ E**−ATP prior to DnaK-catalyzed ATP hydrolysis, where the formation of a collisional complex (E−ATP) causes no change in fluorescence but is followed by two first-order transitions that reduce the fluorescence. (ii) The kinetics of ADP replacement from preformed DnaK−ADP complexes by ATP followed simple exponential kinetics, k ADP = 0.038 (± 0.002) s-1 at 35 °C. The ADP off rate was reduced ∼ 10-fold by inorganic phosphate (20 mM). (iii) Single-turnover experiments ([DnaK] = [ATP] = 1 μM) revealed a slow, first-order increase in tryptophan fluorescence [k obs = 0.0015 (± 0.0001) s-1, 37 °C] that was identical to the rate of DnaK-catalyzed ATP hydrolysis [k hy = 0.0014 (± 0.0001) s-1, 37 °C]. This slow increase in fluorescence is consistent with a E** → E conformational transition. A model for the ATPase cycle of DnaK is proposed in which ATP has two distinct functions: ATP binding to the ATPase domain triggers two conformational transitions in a chaperone molecule, and ATP hydrolysisthe slow step in the reaction cyclereverses the transitions.