Activation of oligopeptidase B from Streptomyces griseus by thiol-reacting reagents is independent of the single reactive cysteine residue.
Oligopeptidase B from Streptomyces griseus was cloned and characterized to clarify the substrate recognition mechanism and the role of a reactive cysteine residue in family S9 prolyl oligopeptidases (POPs). The cloned enzyme, SGR-OpdB, was annotated as a putative family S9 prolyl oligopeptidase based on its deduced amino acid sequence, in which a sole cysteine residue Cys(544) is present close to the catalytic Asp residue in the C-terminal region. The protein was identified as oligopeptidase B, a member of the subfamily S9a of the family S9 POPs, as judged by its substrate specificity and enzymatic characteristics. Its enzymatic activity was markedly enhanced by high NaCl concentration and the reducing reagents dithiothreitol (DTT) and reduced glutathione (GSH). It is particularly interesting that oxidized glutathione (GSSG) also enhanced SGR-OpdB activity. The SGR-OpdB C544A mutant was constructed and characterized to clarify the role of the putative reactive Cys residue, Cys(544). Surprisingly, the enzymatic activity of the Cys-free mutant was also markedly activated by the general thiol-reacting reagent DTT, GSH, and GSSG. To our knowledge, this is the first report of activity-enhancing effects of thiol-reacting reagents toward Cys-free enzymes. Results clarified the role of additives in inducing conformational change of SGR-OpdB into active peptidase.
Usuki
Hirokazu
H
Research Institute for Biological Sciences (RIBS), Okayama, 7549-1 Kibichuo-cho, Kaga-gun, Okayama 716-1241, Japan.
Uesugi
Yoshiko
Y
Iwabuchi
Masaki
M
Hatanaka
Tadashi
T
D000595:Amino Acid Sequence; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation / Q000187:drug effects; D012697:Serine Endopeptidases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D013305:Streptomyces griseus / Q000201:enzymology; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D013439:Sulfhydryl Reagents; D012965:Sodium Chloride; D012697:Serine Endopeptidases; C090703:oligopeptidase B