PMID:9454592

Citation	Slepenkov, SV and Witt, SN (1998) Kinetics of the reactions of the Escherichia coli molecular chaperone DnaK with ATP: evidence that a three-step reaction precedes ATP hydrolysis. Biochemistry 37:1015-24
Abstract	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 degrees 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, kADP = 0.038 (+/- 0.002) s-1 at 35 degrees C. The ADP off rate was reduced approximately 10-fold by inorganic phosphate (20 mM). (iii) Single-turnover experiments ([DnaK] = [ATP] = 1 microM) revealed a slow, first-order increase in tryptophan fluorescence [k(obs) = 0.0015 (+/- 0.0001) s-1, 37 degrees C] that was identical to the rate of DnaK-catalyzed ATP hydrolysis [k(hy) = 0.0014 (+/- 0.0001) s-1, 37 degrees 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.
Links	PubMed Online version:10.1021/bi9720484
Keywords	Adenosine Diphosphate/metabolism; Adenosine Triphosphate/metabolism; Bacterial Proteins/metabolism; Escherichia coli; Escherichia coli Proteins; HSP70 Heat-Shock Proteins/metabolism; Hydrolysis; Kinetics; Models, Chemical; Molecular Chaperones/metabolism; Spectrometry, Fluorescence; Thermodynamics; Tryptophan/chemistry
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