PMID:8526938
| Citation |
Seol, JH, Woo, KM, Kang, MS, Ha, DB and Chung, CH (1995) Requirement of ATP hydrolysis for assembly of ClpA/ClpP complex, the ATP-dependent protease Ti in Escherichia coli. Biochem. Biophys. Res. Commun. 217:41-51 |
|---|---|
| Abstract |
The ATP-dependent protease Ti (Clp) consists of two distinct components, ClpP containing the serine active sites for proteolysis and ClpA having two ATP-binding sites. A ClpA variant (ClpAT) carrying Thr in place of Met169 is highly soluble but indistinguishable from the wild-type ClpA in its ability to hydrolyze ATP and to support the ClpP-mediated proteolysis. Here we show that ATP hydrolysis is essential for assembly of ClpAT/ClpP complex upon analysis of the mixture of its components by gel filtration followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Either ADP or adenosine 5'-(beta,gamma-imido)-triphosphate could not support the complex formation. Furthermore, ClpAT/K501T which carries a mutation in the second ATP-binding site and therefore is unable to cleave ATP could not interact with ClpP. On the other hand, ClpAT/K220T carrying a mutation in the first site and ClpP could be assembled into a complex at 2 mM ATP but not at 0.5 mM, at which concentration the trimeric mutant protein can not form a hexamer. These results indicate that assembly of protease Ti requires hydrolysis of ATP by ClpA in addition to its binding for hexamer formation. |
| Links |
PubMed Online version:10.1006/bbrc.1995.2743 |
| Keywords |
Adenosine Triphosphatases; Adenosine Triphosphate/metabolism; Amino Acid Sequence; Binding Sites; Endopeptidase Clp; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins; Genetic Variation; Hydrolysis; Kinetics; Molecular Sequence Data; Molecular Weight; Point Mutation; Protein Conformation; Serine Endopeptidases/chemistry; Serine Endopeptidases/genetics; Serine Endopeptidases/metabolism |
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