Premium
Mutagenesis of two N‐terminal Thr and five Ser residues in HslV, the proteolytic component of the ATP‐dependent HslVU protease
Author(s) -
Yoo Soon Ji,
Shim Yoon Kyung,
Seong Ihn Sik,
Seol Jae Hong,
Kang Man-Sik,
Chung Chin Ha
Publication year - 1997
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(97)00742-4
Subject(s) - protease , chemistry , mutagenesis , site directed mutagenesis , biochemistry , protein subunit , aaa proteins , proteolysis , atpase , proteasome , enzyme , mutation , mutant , gene
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 β‐type subunit of the 20S proteasomes. To gain an insight into the catalytic mechanism of HslV, site‐directed mutagenesis was performed to replace 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 replaced with Ala. The results obtained by the mutational analysis revealed that the N‐terminal Thr acts as the active site nucleophile and that certain Ser residues, particularly Ser 124 and Ser 172 , also contribute to the peptide hydrolysis by the HslVU protease. The mutational studies also revealed that both Thr, Ser 103 , and Ser 172 , but not Ser 124 , are involved in the interaction of HslV with HslU and hence in the activation of HslU ATPase as well as in the HslVU complex formation.