Kinetic characterization of the ATPase cycle of the DnaK molecular chaperone

Biochemistry
R RussellR McMacken

Abstract

DnaK, the prototype Hsp70 protein of Escherichia coli, functions as a molecular chaperone in protein folding and protein disassembly reactions through cycles of polypeptide 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 degrees C. Hydrolysis of ATP proceeds very slowly with a rate constant (khyd approximately 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 KD approximately 1 nM. ADP binds considerably less tightly than ATP and dissociates from DnaK with a koff of approximately 0.4 min-1 (compared with a koff of approximately 0.008 min-1 for ATP). However, in the presence of physiologically relevant concentrations of...Continue Reading

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