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Mutational analysis of the ATP-binding site in HslU, the ATPase component of HslVU protease in Escherichia coli.

Authors
  • Shin, D H
  • Yoo, S J
  • Shim, Y K
  • Seol, J H
  • Kang, M S
  • Chung, C H
Type
Published Article
Journal
FEBS Letters
Publisher
Wiley (John Wiley & Sons)
Publication Date
Dec 02, 1996
Volume
398
Issue
2-3
Pages
151–154
Identifiers
PMID: 8977096
Source
Medline
License
Unknown

Abstract

HslU is the ATPase component of the ATP-dependent HslVU protease in Escherichia coli. To gain an insight into the structure and function of HslU, site-directed mutagenesis was performed to generate a mutation in the ATP-binding site of the ATPase (i.e., to replace the Lys63 with Thr). Unlike the wild-type HslU, the mutant form (referred to as HslU/K63T) could not hydrolyze ATP or support the ATP-dependent hydrolysis of N-carbobenzoxy-Gly-Gly-Leu-7-amido-4-methyl coumarin by HslV. The wild-type HslU (a mixture of monomer and dimer) formed a multimer containing 6-8 subunits in the presence of either ATP or ADP, indicating that ATP-binding, but not its hydrolysis, is required for oligomerization of HslU. However, HslU/K63T remained as a monomer whether or not the adenine nucleotides were present. Furthermore, ATP or ADP could protect HslU, but not HslU/K63T, from degradation by trypsin. These results suggest that the mutation in the ATP-binding site results in prevention of the binding of the adenine nucleotides to HslU and hence in impairment of both oligomerization and ATPase function of HslU.

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