Mechanism of Soluble Epoxide Hydrolase

Abstract

18O-Labeled epoxides of trans-1,3-diphenylpropene oxide (tDPPO) and cis-9,10-epoxystearic acid were synthesized and used to determine the regioselectivity of sEH. The nucleophilic nature of sEH catalysis was demonstrated by comparing the enzymatic and nonenzymatic hydrolysis products of tDPPO. The results from single turnover experiments with greater or equal molar equivalents of sEH:substrate were consistent with the existence of a stable intermediate formed by a nucleophilic amino acid attacking the epoxide group. Tryptic digestion of sEH previously subjected to multiple turnovers with tDPPO in H 2 18 O resulted in the isolation and purification of a tryptic fragment containing Asp-333. Electrospray mass spectrometry of this fragment conclusively illustrated the incorporation of 18 O. After complete digestion of the latter peptide it was shown that Asp-333 of sEH exhibited an increased mass. The attack by Asp-333 initiates enzymatic activity, leading to the formation of an ␣-hydroxyacyl-enzyme intermediate. Hydrolysis of the acyl-enzyme occurs by the addition of an activated water to the carbonyl carbon of the ester bond, after which the resultant tetrahedral intermediate collapses, yielding the active enzyme and the diol product.Mammalian epoxide hydrolases (E.C. 3.3.2.3) have been implicated in the metabolism of epoxide containing xenobiotics, many of which are believed to be mutagenic and/or carcinogenic (1-3). During the past 20 years the mammalian microsomal epoxide hydrolase (mEH) 1 has received a great deal of attention partly due to its higher selectivity for cyclic and arene oxides (4 -6). A great deal of work has provided a clear picture of the regio-, stereo-, and enantiospecificity of mEH (7-10). Most researchers agree that mEH hydrolyzes the epoxide via an anti (commonly referred to as trans) opening of the oxirane, where attack of the nucleophile usually takes place at the least sterically hindered carbon. These results are substantiated by substituent effects on the rate of hydrolysis as investigated by Dansette et al. (11) and kinetic solvent isotope studies as reported by Armstrong et al. (12). By the use of single turnover experiments in H 2 18 O (13), Lacourciere and Armstrong have also postulated that the hydrolysis of epoxides by mEH proceeds via the intermediary of a nucleophilic amino acid, yielding an acyl-enzyme intermediate, which is hydrolyzed further to release the diol product and the active enzyme. This is in contrast with the previously more accepted hypothesis in which an activated water molecule was thought to be responsible for direct attack on the epoxide.Much less work has been completed on sEH, but the preliminary data closely parallel those for mEH. Because of the critical role of sEHs in the metabolism of xenobiotics and their possible involvement in the biosynthesis of metabolites (14-16, 18), 2 a detailed understanding of the catalytic mechanism of sEH is imperative. The anti opening of epoxides has been demonstrated on a select few substrates along with H 2 18 O studi...

Biochemical Evidence for the Involvement of Tyrosine in Epoxide Activation during the Catalytic Cycle of Epoxide Hydrolase

“…Both mutations of the two tyrosines increased IC 50 values for ureas tested (Table III). Because the epoxide substrates are susceptible to an S N 2 backside attack on the carbon of epoxide ring by nucleophilic Asp 333 (13), the angle and distance of these two tyrosines are suitable to activate epoxide oxygen in concert with nucleophilic attack by Asp Tyr 381 , and the substrate epoxide oxygen modeled into the sEH active site (26) is quite consistent with the preferred stereochemistry of such interactions reflected by the examples from the Cambridge Structural Data base. Epoxide-phenol hydrogen bond stereochemistry is also consistent with the preferred stereochemistry of hydrogen bonds to tyrosines in refined protein structures as outlined by Ippolito et al (39).…”

“…1) (6). The mechanism of murine sEH has been mainly elucidated from a series of experiments utilizing heavy isotopes, protein mass spectrometry, and sitedirected mutagenesis (12,13). They indicated that Asp 333 acts as a catalytic nucleophile and that a water molecule is activated by the nearby His 523 and Asp 495 pair (Fig.…”

Catalysis of potato epoxide hydrolase, StEH1

“…Although heterogeneous, this group of enzymes share features regarding overall structural fold as well as activesite architecture [20]. The enzyme active site contains a proteinderived nucleophilic group (aspartic acid) that has been shown in mammalian epoxide hydrolases to attack an electrophilic carbon of the bound substrate to form an alkylated enzyme intermediate [21,22]. A nearby acid (tyrosine phenol) facilitates ring opening of the oxirane oxygen [12,14].…”

Binding of Alkylurea Inhibitors to Epoxide Hydrolase Implicates Active Site Tyrosines in Substrate Activation

“…The C-terminal domain, or "catalytic domain," contains the active site residues required for epoxide hydrolysis. By analogy with related ␣/␤ hydrolases such as haloalkane dehalogenase (11)(12)(13) and in accord with labeling (14) and site-directed mutagenesis experiments (15,16) , and a hydrogen bond may form between these two residues during catalysis. Given its active site location and absolute conservation among all sequenced epoxide hydrolases, Tyr 465 is a likely general acid that donates a proton to the epoxide oxygen in the first step of catalysis (8,17).…”