Epoxide hydrolases cytosolic

The human cytosolic epoxide hydrolase (cytosolic EH, cEH, also known as soluble EH) has 554 amino acids (Mr 62.3 kDa) and is the product of the EPHX2 gene [49]. Its specific substrate is trans-stilbene oxide, and it appears... 

Cyclic c/5-wteso-epoxides can be asymmetrically hydrolyzed using hepatic epoxide hydrolases to give tran -diols. In this case, the (5)-configurated oxirane carbon atom is preferentially attacked and inverted to yield an (/ ,/ )-diol (Scheme 2.89) [581, 582]. In comparison to the microsomal epoxide hydrolase, cytosolic EH exhibited a lower stereoselectivity. 

Epoxidation by the introduction of an oxygen atom into flunarizine produced l-[bis(4-fluoro-phenyl)methyl] - 4 - [(3-phenyloxiran - 2 - yl)methyl] piperazine (metabolite 2) and epoxide hydration to a diol, 3-[4-[bis(4-fluorophenyl)methyl]-l-piper-azinyl]-l-phenyl-l,2-propanediol (metabolite 10). Lavrijsen et al. (1992) found metabolites formed by epoxidation at the double bond (metabolite 2) and epoxide hydration (metabolite 10) in incubates with subcellular hepatocyte fractions of male and female rats. Metabolites formed by epoxidation and epoxide hydration were not detected in vivo (Meuldermans et al. 1983), probably because the resulting metabolites were metabolised in vivo, much more quickly than in vitro, into secondary metabolites. A diol metabolite, however, was described for the metabolism of l-butyl-4-dimamyl-piperazine in guinea pigs (Morishita et al. 1978). With supernatant fractions a rapid disappearance of the epoxide intermediate from incubate was observed. This seems to indicate that, for the epoxide hydrolysis, besides microsomal epoxide hydrolase, cytosolic epoxide hydrolase might also be involved. 

It should be noted that as early as 1993, Kurth and coworkers investigated the enzymatic transformation of bis-epoxides of type 8-51 using cytosolic epoxide hydrolase from rat liver. However, at that time the regio- and stereochemistry of the obtained THFs had not been investigated. 

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. 

The cytosolic enzyme leukotriene A4 hydrolase (EC 3.3.2.6), which ster-eoselectively converts leukotriene A4 (LTA4) to leukotriene B4 [56], This enzyme catalyzes the hydrolytic cleavage of the 5,6-epoxide ring in LTA4, but, in contrast to what happens with other EHs, the product is not a vicinal diol but a 5,12-diol. As a zinc metalloenzyme, LTA4 hydrolase does not appear to be related to any other epoxide hydrolase. 

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... 

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. 

G. M. Pacifici, A. Temellini, L. Giuliani, A. Rane, H. Thomas, F. Oesch, Cytosolic Epoxide Hydrolase in Humans Development and Tissue Distribution , Arch. Toxicol. 1988, 62, 254 - 257. 

L. Schladt, W. Worner, F. Setiabudi, F. Oesch, Distribution and Inducibility of Cytosolic Epoxide Hydrolase in Male Sprague-Dawley Rats , Biochem. Pharmacol. 1986, 35, 3309 - 3316. 

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. 

E. C. Dietze, J. Stephens, J. Magdalou, D. M. Bender, M. Moyer, B. Fowler, B. D. Hammock, Inhibition of Human and Murine Cytosolic Epoxide Hydrolase by Group-Selective Reagents , Comp. Biochem. Physiol., B 1993, 104, 299 - 308. 

G. Bellucci, C. Chiappe, F. Marioni, M. Benetti, Regio- and Enantioselectivity of the Cytosolic Epoxide Hydrolase-Catalysed Hydrolysis of Racemic Monosubstituted Alkyloxiranes ,./. Chem. Soc., Perkin Trans. 1 1991, 361 - 363 G. Bellucci, C. Chiappe, L. Conti, F. Marioni, G. Pierini, Substrate Enantioselection in the Microsomal Epoxide Hydrolase Catalyzed Hydrolysis of Monosubstituted Oxiranes. Effects of Branching of Alkyl Chains ,./. Org. Chem. 1989, 54, 5978 - 5983. 

N. Chacos, J. Capdevilla, J. R. Falck, S. Manna, C. Martin-Wixtrom, S. S. Gill, B. D. Hammock, R. W. Estabrook, The Reaction of Arachidonic Acid Epoxides (Epoxyeico-satrienoic Acids) with a Cytosolic Epoxide Hydrolase , Arch. Biochem. Biophys. 1983, 223, 639 - 648. 

J. Meijer, J. W. DePierre, Properties of Cytosolic Epoxide Hydrolase Purified from the Liver of Untreated and Clofibrate-Treated Mice , Eur. J. Biochem. 1985, 150,1 - 16. 

J. Magdalou, B. D. Hammock, 1,2-Epoxycycloalkanes Substrates and Inhibiors of Microsomal and Cytosolic Epoxide Hydrolases in Mouse Liver , Biochem. Pharmacol. 1988, 37, 2717 - 2722. 

G. Bellucci, C. Chiappe, F. Marioni, Enantioselectivity of the Enzymatic Hydrolysis of Cyclohexene Oxide and ( )-l-Methylcyclohexene Oxide A Comparison between Microsomal and Cytosolic Epoxide Hydrolases , J. Chem. Soc., Perkin Trans. 1 1989, 2369 -2373. 

Enzyme catalysed hydrolysis of racemic epoxides is interesting from a practical point of view. This reaction is catalysed by epoxide hydrolases (EHs, EC 3.3.2.3) (Archelas and Furstoss, 1998). Mammalian EHs are the most widely studied and they are divided into five groups among which the soluble (cytosolic) epoxide hydrolases (sEH) and microsomal epoxide hydrolases (mEH) are best charactelised. The mechanism of sEH from rat starts with a nucleophilic attack by Asp333 on a carbon of the epoxide (usually the least hindered one) to form a glycol monoester intermediate which is stabilised by an oxyanion hole. A water molecule activated by His523 releases the 1,2-diol product. An... 

Investigations in vitro have demonstrated that epoxybutene is eliminated by microsomal epoxide hydrolase and by cytosolic glutathione V-transferase (GST). Epoxide hydro-... 

Diepoxybutane, like epoxybutene, is eliminated by microsomal epoxide hydrolase in liver and lung of mouse, rat and man (Boogard Bond, 1996) and by cytosolic GST in liver and lung of mouse and rat and in liver of man (Boogard et al., 1996). 

Bentley, P., Bieri, F., Kuslcr. H., Muakkassah-Kelly, S., Sagelsdorff. P, Staubli, W. Waechter, F. (1989) Hydrolysis of bisphenol A diglycidylether by epoxide hydrolases in cytosolic and microsomal fractions of mouse liver and skin inhibition by bis epoxycyclopentylether and the effects upon the covalent binding to mouse skin DNA. Carcinogenesis, 10, 321-327... 

Conversion of arene oxides to dihydrodiols by mouse liver cytosol epoxide hydrolase and microsomal epoxide hydrolase has been compared, and it is found that the former is less active than the latter.200... 

Hydration of epoxides catalyzed by epoxide hydrolase is involved in both detoxication and intoxication reactions. With high concentrations of styrene oxide as a substrate, the relative activity of hepatic microsomal epoxide hydrolase in several animal species is rhesus monkey > human = guinea pig > rabbit > rat > mouse. With some substrates, such as epoxidized lipids, the cytosolic hydrolase may be much more important than the microsomal enzyme. 

Since liver is the most important organ for metabolism investigations the procedures described here focus on liver cytosol exemplarily. Liver cytosol fraction contains soluble Phase I and Phase II enzymes which play an important role in drug metabolism (Brandon 2003). These are alcohol and aldehyde dehydrogenases, epoxide hydrolases, sulfotransferases, glutathione S transferase, N-acetyl transferases, and methyl transferases. Therefore, in cytosolic preparations these biotransformation steps can be studied. Cytosolic fractions are commercially available (BDGentest, Invitro Technologies, Xenotech and others) or easy to prepare, alternatively. 

Epoxide rings of certain alkene and arene compounds are hydrated enzymatically by epoxide hydrolases to form the corresponding iram-dihydrodiols (Figure 10.11). The epoxide hydrolases are a family of enzymes known to exist both in the endoplasmic reticulum and in the cytosol. In earlier studies they were named epoxide hydratase, epoxide hydrase, or epoxide hydrolase. Epoxide hydrolase, however, has been recommended by the International Union of Biochemists Nomenclature Committee and is now in general use. 

Microsomal epoxide hydrolase is widely distributed, having been described from plants, invertabrates, and vertebrates. In vertebrates it has wide organ distribution for example, in the rat, the most studied species, the enzyme has been found in essentially every organ and tissue. Although predominantly located in the endoplasmic reticulum (microsomes), epoxide hydrolase is also found in the plasma and nuclear membranes and, to some extent, in the cytosolic fraction. 

The existence of a cytosolic epoxide hydrolase was first indicated by its ability to hydrolyze analogs of insect juvenile hormone not readily hydrolyzed by microsomal epoxide hydrolase. Subsequent studies demonstrated a unique cytosolic enzyme catalytically and structurally distinct from the microsomal enzyme. It appears probable that the cytosolic enzyme is peroxisomal in origin. Both enzymes are broadly nonspecific and have many substrates in common. It is clear, however, that many substrates hydrolyzed well by cytosolic epoxide hydrolase are hydrolyzed poorly by microsomal epoxide hydrolase and vice versa. For example, l-(4 -ethylphenoxy)-3,7-dimethy I -6,7-epoxy-//7//i,v-2-octene, a substituted geranyl epoxide insect juvenile hormone mimic, is hydrolyzed 10 times more rapidly by the cytosolic enzyme than by the microsomal one. In any series, such as the substituted styrene oxides, the trans configuration is hydrolyzed more rapidly by the cytosolic epoxide hydrolase than is the cis isomer. At the same time, it should remembered that in this and other series,... 

Leukotriene A4 hydrolase is a unique cytosolic epoxide hydrolase, structurally dissimilar to the cytosolic enzyme described above. Its substrate specificity is narrow, being restricted to leukotriene A4, (5(S)-trans-5,6-oxido-7,9-cis-ll,l4-trans-eicosatetraenoic acid), and related fatty acids. 

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