ATP lowers the activation enthalpy barriers to DnaK-peptide complex formation and dissociation.
The role of nucleotide in controlling the pre-steady-state kinetics of peptide binding to the Escherichia coli 70-kDa molecular chaperone DnaK was investigated using stopped-flow fluorescence. The peptide used in this study, fVSV13 (representing amino acids 490-502 of the vesicular stomatitis virus glycoprotein), was dansylated specifically at its N-terminus. We found that (i) between 17 and 35 degrees C in the presence of ATP the second-order rate constant (k(on)) for fVSV13 binding to DnaK exhibited almost no dependence on temperature and did not deviate significantly from 3.8 x 10(5) M(-1) s(-1). In contrast, over the same temperature range in the presence of ADP, k(on) increased by a factor of 32 (7.3 x 10(4) to 2.3 x 10(6) M(-1) s(-1)); and (ii) ATP increased the apparent first-order rate constant for the release of fVSV13 from preformed DnaK-fVSV13 complexes by several orders of magnitude relative to ADP. The activation energy parameters for fVSV13 binding to and dissociation from DnaK are compared to the activation parameters for the binding of an unrelated peptide to DnaK and are also discussed in terms of an open-to-closed equilibrium in the polypeptide-binding domain. On the basis of this comparison, it is suggested that the activation entropy term deltaS++, which is related to the structure of the DnaK-bound peptide or the degree of solvation of the peptide, is a controlling factor in the kinetics of peptide binding to DnaK.
Farr
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Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport 71130, USA.
Witt
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D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D007700:Kinetics; D018832:Molecular Chaperones / Q000378:metabolism*; D011108:Polymers / Q000378:metabolism; D011485:Protein Binding; D014721:Vesicular stomatitis Indiana virus; D014764:Viral Proteins / Q000378:metabolism
D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D011108:Polymers; D014764:Viral Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C063842:dnaK protein, E coli