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

FEBS Letters

Shim, Yoon Kyung¹  Chung, Chin Ha¹  Shin, Dong Hun¹  Kang, Man-Sik¹  Yoo, Soon Ji¹  Seol, Jae Hong¹ 

[1] Department of Molecular Biology and Research Center for Cell Differentiation, College of Natural Sciences, Seoul National University, Seoul 151–742, South Korea

关键词: ATP-dependent protease;    ATPase;    HslVU;    ClpAP;    Escherichia coli;   

DOI  :  10.1016/S0014-5793(96)01223-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown