The effects of mutations in the carboxyl-terminal region on the catalytic activity of Escherichia coli signal peptidase I.
Escherichia coli signal peptidase I (SPase I) is a membrane-bound serine endopeptidase that catalyses the cleavage of signal peptides from the pre-forms of membrane or secretory proteins. Our previous studies using chemical modification and site-directed mutagenesis suggested that Trp(300) and Arg(77), Arg(222), Arg(315) and Arg(318) are important for the proper and stable conformation of the active site of SPase I. Interestingly, many of these residues reside in the C-terminal region of the enzyme. As a continuation of these studies, we investigated in the present study the effects of mutations in the C-terminal region including amino acid residues at positions from 319 to 323 by deletions and site-directed mutagenesis. As a result, the deletion of the C-terminal His(323) was shown to scarcely affect the enzyme activity of SPase I, whereas the deletion of Gly(321)-His(323) or Ile(319)-His(323) as well as the point mutation of Ile(322) to alanine was shown to decrease significantly both the activity in vitro and in vivo without a big gross conformational change in the enzyme. These results suggest a significant contribution of Ile(322) to the construction and maintenance of the proper and critical local conformation backing up the active site of SPase I.
Kim
Yong-Tae
YT
Department of Chemistry, Faculty of Science and Engineering, Aoyama Gakuin University, Sagamihara, Kanagawa, Japan.
Yoshida
Hisashi
H
Kojima
Masaki
M
Kurita
Ryo
R
Nishii
Wataru
W
Muramatsu
Tomonari
T
Ito
Hisashi
H
Park
Sun Joo
SJ
Takahashi
Kenji
K
D001665:Binding Sites; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016296:Mutagenesis; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D008565:Membrane Proteins; D012697:Serine Endopeptidases; C026515:type I signal peptidase