Isolation and characterization of an Escherichia coli DnaK mutant with impaired ATPase activity.
A temperature-sensitive mutant of DnaK, the principal Escherichia coli member of the 70 kDa heat shock protein family, has been isolated. The mutation, dnaK25, lies in the putative ATP binding pocket of DnaK. It consists of a C to T transition that changes the highly conserved proline 143 to serine. Mutant strains do not support the propagation of bacteriophage lambda or of plasmids that require DnaA for replication. They are also defective in the utilization of mannose and sorbitol. ATPase activity of the mutant protein is reduced 20-fold relative to wild-type, while autophosphorylation is unaffected. DnaK25 has a fourfold faster rate of nucleotide exchange than wild-type DnaK; nucleotide exchange by both proteins is markedly increased by GrpE. The DnaK25 ATPase is still stimulated by DnaJ and GrpE and by peptide substrates. However, the affinity of most peptides tested for stimulating the DnaK25 ATPase is reduced significantly. These results indicate that a mutation in the N-terminal nucleotide binding domain can alter substrate interactions with the C-terminal substrate binding site. Nucleotide exchange by both wild-type DnaK and DnaK25 proceeds at a much faster rate than ATP hydrolysis, and therefore cannot be the rate limiting step of ATP hydrolysis under the conditions used in these experiments. Consistent with this, peptides, which stimulate ATP hydrolysis, have no effect on nucleotide exchange. Peptides thus appear to stimulate the ATPase by acting at another step, such as increasing the rate of phosphate bond cleavage.
Burkholder
W F
WF
Institute of Cancer Research, College of Physicians and Surgeons, Columbia University, New York, NY 10032.
Panagiotidis
C A
CA
Silverstein
S J
SJ
Cegielska
A
A
Gottesman
M E
ME
Gaitanaris
G A
GA
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D017422:Sequence Analysis, DNA; D013696:Temperature
D001426:Bacterial Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli