MUTATIONAL ANALYSIS OF THE ATP-BINDING SITE IN HS1U, THE ATPASE COMPONENT OF HS1VU PROTEASE IN ESCHERICHIA-COLI

HslU is the ATPase component of the ATP-dependent HslVU protease in Es cherichia coli, To gain an insight into the structure and function of HslU, site-directed mutagenesis was performed to generate a mutation i n the ATP-binding site of the ATPase (i.e., to replace the Lys(63) wit h Thr). Unlike the wild-type HslU, the mutant form (referred to as Hsl U/K63T) could not hydrolyze ATP or support the ATP-dependent hydrolysi s 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 con taining 6-8 subunits in the presence of either ATP or ADP, indicating that ATP-binding, but not its hydrolysis, is required for oligomerizat ion of HslU, However, HslU/K63T remained as a monomer whether or not t he adenine nucleotides were present, Furthermore, ATP or ADP could pro tect HslU, but not HslU/K63T, from degradation by trypsin, These resul ts suggest that the mutation in the ATP-binding site results in preven tion of the binding of the adenine nucleotides to HslU and hence in im pairment of both oligomerization and ATPase function of HslU.