Mutagenesis of two N-terminal Thr and five Ser residues in HslV, the proteolytic component of the ATP-dependent HslVU 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-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 Ser124 and Ser172, also contribute to the peptide hydrolysis by the HslVU protease. The mutational studies also revealed that both Thr, Ser103, and Ser172, but not Ser124, 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.
Yoo
S J
SJ
Department of Molecular Biology and Research Center for Cell Differentiation, College of Natural Sciences, Seoul National University, South Korea.
Shim
Y K
YK
Seong
I S
IS
Seol
J H
JH
Kang
M S
MS
Chung
C H
CH
D049069:ATP-Dependent Proteases; D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology*; D000595:Amino Acid Sequence; D019281:Dimerization; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D006360:Heat-Shock Proteins; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism; D012694:Serine / Q000235:genetics*; D012697:Serine Endopeptidases; D013329:Structure-Activity Relationship; D013912:Threonine / Q000235:genetics*
D006360:Heat-Shock Proteins; D010455:Peptides; D013912:Threonine; D012694:Serine; D000255:Adenosine Triphosphate; D010450:Endopeptidases; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; D000251:Adenosine Triphosphatases