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 (His^1^0^3, Glu^1^2^8, Gly^1^6^3, Asp^1^6^4, Ser^1^6^5, Gly^1^6^7, Asp^2^6^2, Asp^2^6^6 and His^2^9^2) by site-directed mutagenesis. Among them, Gly^1^6^3, Asp^1^6^4, Ser^1^6^5, and Gly^1^6^7 are the components of a G-D/E-S-A-G motif. We showed that Ser^1^6^5, Asp^2^6^2, and His^2^9^2 are the active-site residues of the enzyme. We also showed that none of the other residues, except for Asp^1^6^4, is critical for the enzymatic activity. The mutation of Asp^1^6^4 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.