Asp(333), Asp(495), and His(523) form the catalytic triad of rat soluble epoxide hydrolase

作者：Arand M^*; Wagner H; Oesch F

来源：JOURNAL OF BIOLOGICAL CHEMISTRY, 1996, 271(8): 4223-4229.

DOI：10.1074/jbc.271.8.4223

On the basis of the sequence similarity between mammalian epoxide hydrolases and bacterial haloalkane dehalogenase reported earlier (Arand, M., Grant, D. F., Beetham, J. K., Friedberg, T., Oesch, F., and Hammock, B. D. (1994) FEES Lett. 338, 251-256; Beetham, J. K, Grant, D., Arand, M., Garbarino, J., Kiyosue, T., Pinot, F., Oesch, F., Bellnap, W R., Shinozaki, It., and Hammock, B. D. (1995) DNA Cell Biol 14, 61-71) we selected candidate amino acid residues for the putative catalytic triad of the rat soluble epoxide hydrolase. The predicted amino acid residues were exchanged by site-directed mutagenesis of the epoxide hydrolase cDNA, followed by the expression of the respective mutant enzymes in Escherichia coli. A total of 25 different mutants were analyzed for their epoxide hydrolase activity toward the model substrate trans-stilbene oxide. In case of impaired catalytic activity of a given mutant, the structural integrity of the recombinant enzyme protein was assessed either by its ability to covalently bind the substrate trans-stilbene oxide or by affinity purification on benzyl thio-Sepharose, using the soluble epoxide hydrolase-specific competitive inhibitor 4-fluorochalcone oxide to release the bound enzyme from the affinity matrix. Of the mutants under investigation, only those with changes in the positions Asp(333), Asp(495) and His(523) were completely inactive toward the model substrate trans-stilbene oxide while retaining the proper protein fold. These amino acids were exactly those previously predicted by sequence alignment. Exchange of the amino acid residues flanking the catalytic nucleophile Asp(333) significantly changed the kinetic properties of the enzyme. Mutation of His(332) to Gln had no apparent effect on the K-m but led to a heavily reduced V-max (5% that of the wild type) of the mutant enzyme, while the exchange of Trp(334) against Phe strongly increased the K-m (7-fold) and also moderately enhanced the V-max (2-fold) of the corresponding mutant. Mutation of Trp(540) apparently had a strong effect on the protein conformation.

• 出版日期1996-2-23