Identification of catalytically essential residues in Escherichia coli esterase by site-directed mutagenesis.
Escherichia coli esterase (EcE) is a member of the hormone-sensitive lipase family. We have analyzed the roles of the conserved residues in this enzyme (His103, Glu128, Gly163, Asp164, Ser165, Gly167, Asp262, Asp266 and His292) by site-directed mutagenesis. Among them, Gly163, Asp164, Ser165, and Gly167 are the components of a G-D/E-S-A-G motif. We showed that Ser165, Asp262, and His292 are the active-site residues of the enzyme. We also showed that none of the other residues, except for Asp164, is critical for the enzymatic activity. The mutation of Asp164 to Ala dramatically reduced the catalytic efficiency of the enzyme by the factor of 10(4) without seriously affecting the substrate binding. This residue is probably structurally important to make the conformation of the active-site functional.
Haruki
M
M
Department of Material and Life Science, Graduate School of Engineering, Osaka University, Suita, Japan.
Oohashi
Y
Y
Mizuguchi
S
S
Matsuo
Y
Y
Morikawa
M
M
Kanaya
S
S
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D004950:Esterases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment
D001426:Bacterial Proteins; D004950:Esterases