The role of Glu196 in the environment around the substrate binding site of leucine aminopeptidase from Streptomyces griseus.
To investigate the role of Glu196 of leucine aminopeptidase from Streptomyces griseus (SGAP) in SGAP activation by calcium and substrate specificity, we constructed E196X SGAP by saturation mutagenesis. Most mutations led to the abrogation of SGAP activation by calcium, and substitution with Lys led to a marked increase in activity toward Asp-p-nitroanilide (pNA) and a decrease in that toward Lys-pNA. A similar result was obtained from the investigation using non-calcium-activated enzyme from Streptomyces septatus (SSAP). These results indicate that Glu196 of SGAP is associated with the environment around the substrate binding site besides its role in SGAP activation by calcium.
Arima
Jiro
J
Research Institute for Biological Sciences (RIBS), Okayama, Kaga-gun, Okayama 716-1241, Japan.
Uesugi
Yoshiko
Y
Uraji
Misugi
M
Iwabuchi
Masaki
M
Hatanaka
Tadashi
T
D019943:Amino Acid Substitution; D000813:Anilides / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D007931:Leucyl Aminopeptidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D016296:Mutagenesis; D017354:Point Mutation; D017433:Protein Structure, Secondary; D013305:Streptomyces griseus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
D000813:Anilides; D001426:Bacterial Proteins; D008024:Ligands; D007931:Leucyl Aminopeptidase; D002118:Calcium