Microsomal hydrolases

Napoli JL, Pacia EB, Salerno GJ (1989) Cholate-independent hydrolysis of all-fraw -retinyl palmitate by rat tissues solubilization of multiple kidney microsomal hydrolases. Arch Biochem Biophys 21 A 192-199... 

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. 

Drugs may also undergo hydrolysis by intestinal esterases (hydrolases), more specifically carboxylesterases (EC 3.1.1.1) in the intestinal lumen and at the brush border membrane [58, 59]. It has been shown that intestinal hydrolase activity in humans was closer to that of the rat than the dog or Caco-2 cells [60]. In these studies, six propranolol ester prodrugs and p-nitrophenylacetate were used as substrates, and the hydrolase activity found was ranked in the order human > rat Caco-2 cells > dog for intestinal microsomes. The rank order in hydrolase activity for the intestinal cytosolic fraction was rat > Caco-2 cells = human > dog. The hydrolase activity towards p-nitrophenylacetate and tenofovir disoproxil has also been reported in various intestinal segments from rats, pigs and humans. The enzyme activity in intestinal homogenates was found to be both site-specific (duodenum > jejunum > ileum > colon) and species-dependent (rat > man > Pig)-... 

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). 

Pure human CESs (hCEl and hCE2), a rabbit CES (rCE), and two rat CESs (Hydrolases A and B) were used to study the hydrolytic metabolism of the following pyrethroids lR-trans-resmethrin (bioresmethrin), 1RS-1runs-permethrin, and 1RS-r/.v-pennethrin [28], hCEl and hCE2 hydrolyzed /ram-pemiethrin 8- and 28-fold more efficiently than m-permethrin (when kcat/Km values were compared), respectively. In contrast, hydrolysis of bioresmethrin was catalyzed efficiently by hCEl, but not by hCE2. The kinetic parameters for the pure rat and rabbit CESs were qualitatively similar to the human CESs when hydrolysis rates of the investigated pyrethroids were evaluated. Further, a comparison of pyrethroid hydrolysis by hepatic microsomes from rats, mice, and humans indicated that the rates for each compound were similar between species. 

NADPH-independent hydrolytic metabolism. The CLint for deltamethrin was estimated to be twice as rapid in humans as in rats on a per kg body weight basis. Metabolism by purified rat and human CESs was used to examine further the species differences in hydrolysis of deltamethrin and esfenvalerate. Results of CES metabolism revealed that hCEl was markedly more active toward deltamethrin than the Class I rat CESs, hydrolase A and B, and the Class II human CES, hCE2 however, hydrolase A metabolized esfenvalerate twice as fast as hCEl, whereas hydrolase B and hCEl hydrolyzed esfenvalerate at equal rates. These studies demonstrated a significant species difference in the in vitro pathways of biotransformation of deltamethrin in rat and human liver microsomes, which was due in part to differences in the intrinsic activities of rat and human CESs. 

Three isoenzymes of carboxylesterase were purified from rat liver micro-somes and were named RL1, RL2, and RH1. These differ from each other in their response to hormone treatment, inducibility, substrate specificity, and immunological properties [75], It was shown that RL1, RL2, and RH1 resemble hydrolases p/ 6.2/6.4, pI 6.0, and pI 5.6, respectively. Enzyme RL2 was found to be identical to egasyn, a protein with esterase activity found in the endoplasmic reticulum [76], The role of egasyn is to stabilize glucuronidase (EC 3.2.1.31) by noncovalent binding to the microsomal membrane. 

Thiolester hydrolases are present in most tissues and cell compartments. High concentrations are found in liver microsomes and in brown adipose tissue mitochondria and peroxisomes. Several acyl-CoA hydrolases have shown a close relationship to the nonspecific carboxylesterases EC 3.1.1.1. Thus, palmitoyl-CoA hydrolase purified from rat liver microsomes was found to be identical to esterase pI 6.2I6A (ES4 type). An acyl-CoA hydrolase was isolated that showed high similarity to esterase pI 6.1 [74a] [129] [130]. These few examples are further illustrations of the unsatisfying situation of the traditional classification of esterases. 

M. Robbi, H. Beaufay, Cloning and Sequencing of Liver Carboxylesterase ES3 (Egasyn) , Biochem. Biophys. Res. Commun. 1994, 203, 1404-1411 M. Robbi, E. Van Schaftingen, H. Beaufay, Cloning and Sequencing of Rat Liver Carboxylesterase ES-4 (Microsomal Palmitoyl CoA Hydrolase) , Biochem. J. 1996, 313(Part 3), 821-826. 

R. Verger, Purification and Characterization of a Porcine Liver Microsomal Triacyl-glycerol Hydrolase , Biochemistry 1997, 36, 1861-1868. 

L. Luan, T. Sugiyama, S. Takai, Y. Usami, T. Adachi, Y. Katagiri, K. Hirano, Purification and Characterisation of Pranlukast Hydrolase from Rat Liver Microsomes The Hydrolase Is Identical to Carboxylesterase p/ 6.2 , Biol. Pharm. Bull. 1997, 20, 71-75. 

In comprehensive studies, the hydrolysis of some 30 naphthyl esters by human, rat, and mouse liver carboxylesterases was investigated [43], A general trend that was apparent was that the rate of hydrolysis of a- and /3-naphthyl carbonates (7.21, R = alkyl or arylalkyl) catalyzed by human microsomes or rat hydrolases showed a tendency to decrease with increasing lipophilic-ity (range ca. 2 to 5). A similar trend was not seen with naphthyl aryl carbonates nor with a-naphthyl carboxylates. These results tell us that, even with purified enzymes and large series of substrates, it is very difficult indeed to discern sound structure-hydrolysis relationships due to the complexity of the structural and enzymatic factors involved. 

The transesterification of cocaine to cocaethylene is an enzymatic reaction catalyzed by microsomal carboxylesterases and blocked by inhibitors of serine hydrolases [124][125], In Chapt. 3, we have discussed the mechanism of serine hydrolases, showing how a H20 molecule enters the catalytic cycle to hydrolyze the acylated serine residue in the active site of the enzyme. In the case of cocaine, the acyl group is the benzoylecgoninyl moiety (Fig. 7.9,d ), which undergoes esterification with ethanol according to Steps e and/ (Fig. 7.9). 

R. P. Lee, A. Parkinson, P. G. Forkert, Isozyme-Selective Metabobsm of Ethyl Carbamate by Cytochrome P450 (CYP2E1) and Carboxylesterase (Hydrolase A) Enzymes in Murine Liver Microsomes , Drug Metab. Dispos. 1998, 26, 60-65. 

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 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]). 

Amino acid sequence relationships have suggested a number of HYL families based on percent identity, enzymes with >40% identity belonging to the same family [48]. Families so identified include the mammalian microsomal EH (HYL1), the mammalian cytosolic EH (HYL2), the plant cytosolic EH (HYL3), and bacterial C-X bond hydrolases (haloacid dehydrogenases, HAD, and haloalkane dehalogenases, HLD). 

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. 

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