MUTAGENESIS OF 2 N-TERMINAL THR AND 5 SER RESIDUES IN HS1V, THE PROTEOLYTIC COMPONENT OF THE ATP-DEPENDENT HS1VU PROTEASE

HslVU in E. coli is a new type of ATP-dependent protease consisting of two heat shock proteins: the HslU ATPase and the HslV peptidase that has two repeated Thr residues at its N terminus, like certain beta-typ e subunit of the 20S proteasomes, To gain an insight into the catalyti c mechanism of HslV, site-directed mutagenesis was performed to replac e each of the Thr residues with Ser or Val and to delete the first or both Thr, Also each of the five internal Ser residues in HslV were rep laced with Ala, The results obtained by the mutational analysis reveal ed that the N-terminal Thr acts as the active site nucleophile and tha t certain Ser residues, particularly Ser(124) and Ser(172), also contr ibute to the peptide hydrolysis by the HslVU protease, The mutational studies also revealed that both Thr, Ser(103), and Ser(172), but not S er(124), are involved in the interaction of HslV with HslU and hence i n the activation of HslU ATPase as well as in the HslVU complex format ion. (C) 1997 Federation of European Biochemical Societies.