Identification of catalytically essential residues in Escherichia coli esterase by site-directed mutagenesis

作者：Haruki M; Oohashi Y; Mizuguchi S; Matsuo Y; Morikawa M; Kanaya S^*

来源：FEBS Letters, 1999, 454(3): 262-266.

DOI：10.1016/S0014-5793(99)00813-3

• site-directed mutagenesis
• active site
• hormone-sensitive lipase
• esterase
• Escherichia coli

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(103), Glu(128), Gly(163), Asp(164), Ser(165), Gly(167), Asp(262), Asp(266) and His(292)) by site-directed mutagenesis. Among them, Gly(163), Asp(164), Ser(165), and Gly(167) are the components of a G-D/E-S-A-G motif. We showed that Ser(165), Asp(262), and His(292) are the active-site residues of the enzyme. We also showed that none of the other residues, except for Asp(164), is critical for the enzymatic activity. The mutation of Asp(164) 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.

• 出版日期1999-7-9