Microsomal epoxide hydrolases

The microsomal epoxide hydrolases (microsomal EH, mEH), predominantly found in the endoplasmic reticulum, regio- and stereoselectively catalyze the hydration of both alkene and arene oxides, including oxides of polycyclic aromatic hydrocarbons. These enzymes have been purified to homogeneity from various species and tissues [22] [41 - 46], The human microsomal EH contains 455 amino acids (Mr 52.5 kDa) and is the product of the EPHX1 gene [47] (also known as HYL1 [48]). 

Water conjugation Water Epoxide hydrolase (microsomes) Arene oxides, cis-disubstituted and mono-substituted oxiranes Benzopyrene 7,8-epoxide, styrene 1,2-oxide, carbamazepine epoxide... 

NAD (P) H quinone oxidoreductase (122) + Glucose-6-phosphate dehydrogenase (123) + Epoxide hydrolase, microsomal (124)... 

The microsomal fraction consists mainly of vesicles (microsomes) derived from the endoplasmic reticulum (smooth and rough). It contains cytochrome P450 and NADPH/cytochrome P450 reductase (collectively the microsomal monooxygenase system), carboxylesterases, A-esterases, epoxide hydrolases, glucuronyl transferases, and other enzymes that metabolize xenobiotics. The 105,000 g supernatant contains soluble enzymes such as glutathione-5-trans-ferases, sulfotransferases, and certain esterases. The 11,000 g supernatant contains all of the types of enzyme listed earlier. 

Lewis DF, Lake BG, Bird MG. Molecular modelling of human microsomal epoxide hydrolase (EH) by homology with a fungal (Aspergillus niger) EH crystal structure of 1.8 A resolution structure-activity relationships in epoxides inhibiting EH activity. Toxicol In Vitro 2005 19 517-22. 

Parkinson, A., Thomas, P.E., Ryan, D.E. etal. (1983) Differential time course of induction of rat liver microsomal cytochrome P 450 isozymes and epoxide hydrolase by Aroclor 1254. Archives of Biochemistry and Biophysics, 225, 203-215. 

Figure 1. The major pathways in the metabolism of BaP to BaP epoxides, dihydrodiol, and 7,8-dihydrodiol-9,10-epoxides. The absolute configurations are as shown. The position of trans-addition of water is shown by an arrow. The optical purity of the 4,5-epoxide formed in BaP metabolism is dependent on the cytochrome P-450 isozymes present in the microsomal enzyme system. EH epoxide hydrolase.

It was recently reported that. >97% of BaP 4,5-epoxide metabolically formed from the metabolism of BaP in a reconstituted enzyme system containing purified cytochrome P-450c (P-448) is the 4S,5R enantiomer (24). The epoxide was determined by formation, separation and quantification of the diastereomeric trans-addition products of glutathione. Recently a BaP 4,5-epoxide was isolated from a metabolite mixture obtained from the metabolism of BaP by liver microsomes from 3-methylcholanthrene-treated Sprague-Dawley rats in the presence of the epoxide hydrolase inhibitor 3,3,3-trichloropropylene oxide, and was found to contain a 4S,5R/4R,5S enantiomer ratio of 94 6 (Chiu et. al., unpublished results). However, the content of the 4S,5R enantiomer was <60% when liver microsomes from untreated and phenobarbital-treated rats were used as the enzyme sources. Because BaP 4R,5S-epoxide is also hydrated predominantly to 4R,5R-dihydro-... 

It was shown that microsomal epoxide hydrolase-catalyzed trans-addition of water to BaP 9,10-epoxide occurs stereospecifically at the C-9 position (15). Since BaP is metabolized essentially to an optically pure 9R,10R-dihydrodiol (13 and L5 Table I), the 9,10-epoxide formed in BaP metabolism must have 9S,10R absolute stereochemistry (Figure 1). Similarly, the 7,8-epoxide formed in BaP metabolism is hydrated specifically at the C-8 position to form the 7R,8R-dihydrodiol (14.21). Hence the enzymatically formed 7,8-epoxide intermediate has 7R,8S absolute stereochemistry (Figure 1). Although the 7R,8R-dihydrodiol is formed almost exclusively from BaP metabolism in rat liver microsomes (Table I) and in bovine bronchial explants (25). the 7S,8S-dihydrodiol is also formed from BaP metabolism in mouse skin epidermis in vivo (5). 

In contrast to the metabolism of BA and BaP, the 5,6-dihydrodiols formed in the metabolism of DMBA by liver microsomes from untreated, phenobarbital-treated, and 3-methylcholanthrene-treated rats are found to have 5R,6R/5S,6S enantiomer ratios of 11 89, 6 94, and 5 95, respectively (7.49 and Table II). The enantiomeric contents of the dihydrodiols were determined by a CSP-HPLC method (7.43). The 5,6-epoxide formed in the metabolism of DMBA by liver microsomes from 3MC-treated rats was found to contain predominantly (>97%) the 5R,6S-enantiomer which is converted by microsomal epoxide hydrolase-catalyzed hydration predominantly (>95%) at the R-center (C-5 position, see Figure 3) to yield the 5S,6S-dihydrodiol (49). In the metabolism of 12-methyl-BA, the 5S,6S-dihydrodiol was also found to be the major enantiomer formed (50) and this stereoselective reaction is similar to the reactions catalyzed by rat liver microsomes prepared with different enzyme inducers (unpublished results). Labeling studies using molecular oxygen-18 indicate that 5R,68-epoxide is the precursor of the 5S,6S-dihydrodiol formed in the metabolism of 12-methyl-BA (51). 

The 8,9- and 10,11-dihydrodiols formed in the metabolism of BA and DMBA respectively are all highly enriched (>90%) in R,R enantiomers (Table III). Labeling experiments using molecular oxygen-18 in the in vitro metabolism of the respective parent compounds and subsequent mass spectral analyses of dihydrodiol metabolites and their acid-catalyzed dehydration products indicated that microsomal epoxide hydrolase-catalyzed hydration reactions occurred exclusively at the nonbenzylic carbons of the metabolically formed epoxide intermediates (unpublished results). These findings indicate that the 8,9- and 10,11-epoxide intermediates, formed in the metabolism of BA and DMBA respectively, contain predominantly the 8R,9S and 10S,11R enantiomer, respectively. These stereoselective epoxidation reactions are relatively insensitive to the cytochrome P-450 isozyme contents of different rat liver microsomal preparations (Table III). 

According to biochemical separation, location, and substrate specificity, epoxide hydrolases (EH) have been divided into a number of groups. In mammals, the insoluble microsomal epoxide hydrolases and the soluble cytosolic epoxide hydrolases are enzymes of broad and complementary substrate specificity. 

From the above, it is clear that the epoxide hydrolases of greatest significance in drug and xenobiotic metabolism are the microsomal and soluble ones. Their catalytic mechanism, which we now examine, is different from that of cholesterol epoxide hydrolase and LTA4 hydrolase (e.g., [57][58]). 

A critical input in unraveling the catalytic mechanism of epoxide hydrolases has come from the identification of essential residues by a variety of techniques such as analysis of amino acid sequence relationships with other hydrolases, functional studies of site-directed mutated enzymes, and X-ray protein crystallography (e.g., [48][53][68 - 74]). As schematized in Fig. 10.6, the reaction mechanism of microsomal EH and cytosolic EH involves a catalytic triad consisting of a nucleophile, a general base, and a charge relay acid, in close analogy to many other hydrolases (see Chapt. 3). 

Fig. 10.6. Simplified representation of the postulated catalytic cycle of microsomal and cytosolic epoxide hydrolases, showing the roles played by the catalytic triad (i.e., nucleophile, general base, and charge relay acid) and some other residues, a) Nucleophilic attack of the substrate to form a /3-hydroxyalkyl ester intermediate, b) Nucleophilic attack of the /Thydroxyal-kyl ester by an activated H20 molecule, c) Tetrahedral transition state in the hydrolysis of the /f-hydroxyalkyl ester, d) Product liberation, with the enzyme poised for a further catalytic... 

Together with glutathione conjugation, hydration is a major pathway in the inactivation and detoxification of arene oxides. Exceptions to this rule will be treated when discussing polycyclic aromatic hydrocarbons. Arene oxides are good substrates for microsomal EH, as evidenced in Table 10.1, where hydration of selected arene oxides, alkene oxides, and cy-cloalkene oxides by purified rat liver epoxide hydrolase is compared. The hy- ... 

Table 10.1. Relative Activity of Rat Liver Microsomal Epoxide Hydrolase toward Some ...

Table 10.2. Epoxide Hydrolase Activity in Human Liver Microsomes [119]... 

An unusual case of intramolecular competition (chemoselectivity, see Chapt. 1 in [la]) between ester and oxirane occurs in the detoxification of (oxiran-2-yl)methyl 2-ethyl-2,5-dimethylhexanoate (10.49), one of the most abundant isomers of an epoxy resin. The compound is chemically very stable, i.e., resistant to aqueous hydrolysis, but is rapidly hydrolyzed in cytosolic and microsomal preparations by epoxide hydrolase and carboxylesterase, which attack the epoxide and ester groups, respectively [129], The rate of overall enzymatic hydrolysis was species dependent, decreasing in the order mouse > rat > human, but was relatively fast in all tissues examined (lung and skin as portals of entry, and liver as a further barrier). In mouse and rat lung microsomes, ester hydrolysis was 3-4 times faster than epoxide hydration, whereas the opposite was true in human lung microsomes. 

Both the mono- and diepoxides of butadiene are substrates for epoxide hydrolase [163], In rat liver microsomes, (R)- and (S)-butadiene monoepoxides were hydrolyzed to but-3-ene-l,2-diol (10.104, Fig. 10.24) with complete retention of configuration at C(2), indicating attack at C(l) [164], In mouse liver microsomes, in contrast, 15 - 25% inversion of configuration was observed, suggesting partial attack at C(2). Preliminary results indicate that human liver microsomes are more efficient than mouse or rat liver microsomes in hydrolyzing butadiene monoepoxide [165]. The hydrolysis of diepoxybutane (10.103) yields 3,4-epoxybutan-l,2-diol (10.105), which can be further hydrated to erytritol (10.106) [163]. 

A. Gaedigk, J. S. Leeder, D. M. Grant, Tissue-Specific Expression and Alternative Splicing of Human Microsomal Epoxide Hydrolase , DNA Cell Biol. 1997, 16, 1257 -1266. 

F. Waechter, P. Bentley, F. Bieri, S. Muakkassah-Kelly, W. Staubli, M. Villermain, Organ Distribution of Epoxide Hydrolases in Cytosolic and Microsomal Fractions of Normal and Nafenopin-Treated Male DBA/2 Mice , Biochem. Pharmacol. 1988, 37, 3897 -3903. 

T. M. Guenthner, D. Cai, R. Wallin, Co-Purification of Microsomal Epoxide Hydrolase with the Warfarin-Sensitive Vitamin Kx Oxide Reductase of the Vitamin K Cycle , Biochem. Pharmacol. 1998, 55, 169 - 175. 

S. Benhamou, M. Reinikainen, C. Bouchardy, P. Dayer, A. Hirvonon, Association between Lung Cancer and Microsomal Epoxide Hydrolase Genotypes , Cancer Res. 1998, 58, 5291 - 5293. 

J. G. Hengstler, M. Arand, M. E. Herrero, F. Oesch, Polymorphism of A-Acetyltransfe-rase, Glutathione S-Transferases, Microsomal Epoxide Hydrolase and Sulfotransferase Influence on Cancer Susceptibility , Recent Results Cancer Res. 1998, 154, 47 - 85. 

T. Friedberg, R. Holler, B. Lollmann, M. Arand, F. Oesch, The Catalytic Activity of the Endoplasmic Reticulum-Resident Protein Microsomal Epoxide Hydrolase towards Carcinogens Is Retained on Inversion of Its Membrane Topology , Biochem. J. 1996, 319, 131 - 136. 

J. Seidegard, J. W. DePierre, Microsomal Epoxide Hydrolase. Properties, Regulation and Function , Biochim. Biophys. Acta 1983, 695, 251 - 270. 

R. C. Skoda, A. Demierre, O. W. McBride, F. J. Gonzalez, U. A. Meyer, Human Microsomal Xenobiotic Epoxide Hydrolase. Complementary DNA Sequence, Complementary DNA-Directed Expression in COS-1 Cells and Chromosomal Localization , J. Biol. Chem. 1988, 263, 1549 - 1554. 

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