Semifunctional Site-Specific Mutants Affecting the Hydrolytic Half-Reaction of Microsomal Epoxide Hydrolase

Abstract

Microsomal epoxide hydrolase (MEH) is a member of the alpha/beta-hydrolase fold family of enzymes, each of which has a catalytic triad consisting of a nucleophile involved in the formation of a covalent intermediate and a general base and charge relay carboxylate that catalyze the hydrolysis of the intermediate. The rate-limiting step in the catalytic mechanism of MEH is hydrolysis of the ester intermediate. An efficient bacterial expression system for a C-terminal hexahistidine tagged version of the native enzyme, which facilitates the isolation of mutant enzymes in which residues involved in the hydrolytic half-reaction have been altered, is described. The H431S mutant of this enzyme is efficiently alkylated by substrate to form the ester intermediate but is unable to hydrolyze the ester to complete the catalytic cycle, a fact that confirms that H431 acts as the base in the hydrolytic half-reaction. The charge relay carboxylate, which is not apparent in paired sequence alignments with other alpha/beta-hydrolase fold enzymes, is thought to be located between residues 340 and 405. A mutagenic survey of all eight Asp and Glu residues in this region reveals that only two (E376 and E404) influence the catalytic mechanism. Steady-state and pre-steady-state kinetic analyses of these residues suggest that both E404 and E376 may serve the charge relay function in the hydrolysis half-reaction. Finally, the tryptophan residue (W150), which resides in the oxyanion hole sequence HGWP, is demonstrated to contribute to the large change in intrinsic protein fluorescence observed when the enzyme is alkylated.

Reaction Mechanism of Fluoroacetate Dehalogenase from Moraxella sp. B

“…Reaction of Fluoroacetate Dehalogenase in H 2 18 O and Digestion of 18 O-Labeled Enzyme with Trypsin-The wild-type fluoroacetate dehalogenase (10 nmol) was lyophilized. The dried enzyme was dissolved in 50 l of H 2 18 O containing 1 mol of sodium fluoroacetate and 20 mol of Tris sulfate (pH 9.5), and the mixture was incubated at 30°C for 12 h. Fluoroacetate was omitted in a control experiment.…”

“…1A, we can expect that the carboxyl group of the residue is labeled with 18 O when the enzymatic defluorination of fluoroacetate is carried out in H 2 18 O. Therefore, we performed the reaction in H 2 18 O as described above.…”

“…1A) is similar to that of haloalkane dehalogenase from X. autotrophicus GJ10 as shown by x-ray crystallography: an aspartate residue at the active site acts as a nucleophile causing the release of a halide ion and the formation of an ester intermediate, which is subsequently hydrolyzed by a water molecule activated by a histidine residue (16). Recently the reaction catalyzed by rat liver microsomal epoxide hydrolase (EC 3.3.2.3) has also been shown to proceed via an ester intermediate: and His-431 act as a nucleophile and a base, respectively (17,18). The amino acid sequence of fluoroacetate dehalogenase from Moraxella sp.…”

Catalysis of potato epoxide hydrolase, StEH1

“…In addition, the positive charge of a protonated imidazole is expected to stabilize a formed hydroxyl ion as well as a negatively charged transition state within the apolar environment of the enzyme active site. Interestingly, when the corresponding catalytic histidine was mutated in the epoxide hydrolase from rat microsomes, only the hydrolytic half-reaction was shown to be affected whereas the alkylation rate was retained [38].…”

Detoxication Strategy of Epoxide Hydrolase—The Basis for a Novel Threshold for Definable Genotoxic Carcinogens

“…However, even this is not sufficient to account for its role as a rapid detoxifier. Recent findings have led to a detailed understanding of the enzymatic mechanism by which mEH and the related soluble epoxide hydrolase hydrolyze their substrates Armstrong, 1993, 1994;Arand et al, 1994Arand et al, , 1996Hammock et al, 1994;Tzeng et al, 1996Tzeng et al, , 1998Müller et al, 1997;Laughlin et al, 1998;Arand et al, in press). These enzymes belong to the large structural family of α/β hydrolase fold enzymes (Ollis et al, 1992).…”