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J Biol Chem, Vol. 273, Issue 36, 22929-22935, September 4, 1998

Effects of the Cys Mutations on Structure and Function of the ATP-dependent HslVU Protease in Escherichia coli
THE CYS²⁸⁷ TO VAL MUTATION IN HslU UNCOUPLES THE ATP-DEPENDENT PROTEOLYSIS BY HslVU FROM ATP HYDROLYSIS

Soon Ji Yoo, Hyun Hee Kim, Dong Hun Shin, Cheol Soon Lee, Ihn Sik Seong, Jae Hong Seol, Naoki Shimbara^¶, Keiji Tanaka, and Chin Ha Chung

From the  Department of Molecular Biology and Research Center for Cell Differentiation, College of Natural Sciences, Seoul National University, Seoul 151-742, Korea, the ^¶ Biomedical R & D Department, Sumitomo Electric Industries, Yokohama 244, Japan, and the  Tokyo Metropolitan Institute of Medical Science, CREST, Japan Science and Technology Corporation, Tokyo 113, Japan

To define the role of the Cys residues in the ATP-dependent HslVU protease, mutagenesis was performed to replace either Cys²⁶¹ or Cys²⁸⁷ in HslU with Val and Cys¹⁵⁹ in HslV with Ser or Ala. Whereas HslU/C261V could hydrolyze ATP and support the ATP-dependent proteolytic activity of HslV as well as the wild-type HslU, HslU/C287V could not hydrolyze ATP. Nevertheless, HslU/C287V could support the HslV-mediated proteolysis by forming the HslVU complex in the presence of ATP but not its absence, indicating that ATP binding but not its hydrolysis is essential for proteolysis. Whereas treatment of N-ethylmaleimide (NEM) resulted in dissociation of the oligomeric HslU into monomers, the C261V mutation, but not C287V, prevented the NEM effect. These results suggest that Cys²⁶¹ is involved in oligomerization and that Cys²⁸⁷ is related to the ATPase function of HslU. NEM also dissociated the dodecameric HslV into monomers, and this effect could be prevented by either the C159S or C159A mutation, suggesting the involvement of Cys¹⁵⁹ in oligomerization of HslV. Moreover, either mutation abolished both the basal and HslU-activated proteolytic activity of HslV and its ability to activate the HslU ATPase and to form the HslVU complex, indicating that Cys¹⁵⁹ is essential for the proteolytic activity of HslV and its interaction with HslU. These results suggest that the Cys residues play an important role in maintaining the structure and function of the HslVU protease.

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