Mechanism of Microsomal Epoxide Hydrolase. Semifunctional Site-Specific Mutants Affecting the Alkylation Half-Reaction

Abstract

Microsomal epoxide hydrolase (MEH) catalyzes the addition of water to epoxides in a two-step reaction involving initial attack of an active site carboxylate on the oxirane to give an ester intermediate followed by hydrolysis of the ester. An efficient bacterial expression system for the enzyme from rat that facilitates the production of native and mutant enzymes for mechanistic analysis is described. Pre-steady-state kinetics of the native enzyme toward glycidyl-4-nitrobenzoates, 1, indicate the rate-limiting step in the reaction is hydrolysis of the alkyl-enzyme intermediate. The enzyme is enantioselective, turning over (2R)-1 about 10-fold more efficiently than (2S)-1, and regiospecific toward both substrates with exclusive attack at the least hindered oxirane carbon. Facile isomerization of the monoglyceride product is observed and complicates the regiochemical analysis. The D226E and D226N mutants of the protein are catalytically inactive, behavior that is consistent with the role of D226 as the active-site nucleophile as suggested by sequence alignments with other alpha/beta-hydrolase fold enzymes. The D226N mutant undergoes hydrolytic autoactivation with a half-life of 9.3 days at 37 degreesC, suggesting that the mutant is still capable of catalyzing the hydrolytic half-reaction (in this instance an amidase reaction) and confirming that D226 is in the active site. The indoylyl side chain of W227, which is in or near the active site, is not required for efficient alkylation of the enzyme or for hydrolysis of the intermediate. However, the W227F mutant does exhibit altered stereoselectivity toward (2R)-1, (2S)-1, and phenanthrene-9,10-oxide, suggesting that modifications at this position might be used to manipulate the stereo- and regioselectivity of the enzyme.

Discovery of a Novel Microsomal Epoxide Hydrolase-Catalyzed Hydration of a Spiro Oxetane

“…EHs are ubiquitous and the hydration of epoxide substrates is energetically favored, with water as the only cosubstrate (Lacourciere and Armstrong, 1993;Hammock et al, 1994;Müller et al, 1997;Laughlin et al, 1998). In mammals, there are several EHs, including sEH, mEH, cholesterol EH, hepoxilin hydrolase, and leukotriene A4 EH (Kodani and Hammock, 2015).…”

“…Based on the established mechanism of EH-catalyzed hydration (Lacourciere and Armstrong, 1993;Tzeng, 1996;Laughlin et al, 1998;Armstrong, 1999), a modified pathway for the mEH-catalyzed hydration of the oxetanyl moiety in AZD1979 is proposed (Fig. 9).…”

Epoxide hydrolases: biochemistry and molecular biology

“…Like sEH, mEH is a member of the a/b-hydrolase enzyme family [66]. Based on its similarity with bacterial haloalkane dehalogenases and other epoxide hydrolases, a catalytic triad consisting of a His 431 , Asp 226 , and a Glu 404 has been proposed [129,130]. These findings are based on earlier reports of a His at position 431 that is essential for catalysis [131], and sequence alignments of related epoxide hydrolases.…”

Catalysis of potato epoxide hydrolase, StEH1

“…A suggested physiological role of the plant enzymes is through their involvement in the epoxide fatty acid metabolism and biosynthetic pathway of cutin [9]. The reaction mechanism of epoxide hydrolases from mammalian [10][11][12] and bacterial [13,14] sources have been thoroughly investigated through mutagenesis studies, kinetic analyses and solved threedimensional structures of enzyme representatives from mouse [15], human [16], Agrobacterium radiobacter AD1 [17] and Aspergillus niger [18] (see [19] for a recent review). The canonical epoxide hydrolases belong to the structural class of α/β hydrolase fold enzymes.…”