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

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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 Lys 63 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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Shin, D. H., Yoo, S. J., Shim, Y. K., Seol, J. H., Kang, M. S., & Chung, C. H. (1996). Mutational analysis of the ATP-binding site in HslU, the ATPase component of HslVU protease in Escherichia coli. FEBS Letters, 398(2–3), 151–154. https://doi.org/10.1016/S0014-5793(96)01223-9

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