Epoxide hydratase activity

Epoxide hydratase activity, with JH-benzo(a)pyrene 4,5-oxide as substrate, was assayed by the thin-layer chromatographic procedure of Jerina et al. (15). The protein content of microsomal and whole homogenate preparations was determined according to Lowry et al. (16), using bovine serum albumin as the standard, and microsomal cytochrome P-450 content was assayed by the method of Omura and Sato (17) on an Aminco DW-2A spectrophotometer. 

The elution profile of cytochrome P-448 (absorption at 418 nm) and epoxide hydratase activity from a sodium cholate-solubi-lized hepatic microsomal preparation (from DBA-treated male skates) applied to a DEAE-cellulose column and eluted with Buffer II is shown in Fig. 3. The void volume of the column contained significant amounts of epoxide hydratase activity. Fractions 40-70 (Fig. 3) were combined, and concentrated. The carbon monoxide difference spectrum, which had an absorption maximum at 448 nm in the induced state, is shown in Fig. 4. This form of the cytochrome (i.e.,... 

Figure 3. Elution profile of partially purified Cytochrome P-448 and epoxide hydratase activity of solubilized hepatic microsomes from DBA-treated male skates from a DEAE-cellulose column with Buffer II. Epoxide hydratase activity was determined with BP-4,5-oxide as the substrate (see Materials and Methods).

When all of the material absorbing at 418 nm (associated with the cytochrome P-448 fractions) was eluted from the DEAE-cellulose column (which in some experiments required more than 1 liter of Buffer II), elution was continued with a linear KC1 gradient (0-0.5 M) in Buffer II, as shown in Fig. 5. A different form(s) of cytochrome P-450 (fractions 130-155), having maximal absorption near 451 nm in the carbon monoxide ligated and reduced form (Fig. 6), was obtained although only 2- to 3-fold purification, relative to microsomes, was achieved. This form of cytochrome P-450 was extensively contaminated with epoxide hydratase activity. However, by combining fractions 130-150 (Fig. 5), it was possible to obtain cytochrome P-451 essentially free of cytochrome b5. The relative content of cytochrome P-448 and cytochrome P-451 Tn the DEAE-column eluates ranged from 1 1.1 to 1 1.6 in several different experiments. 

Other enzymes associated with xenobiotic metabolism are also altered by dietary protein levels. Epoxide hydratase can hydrolyze various epoxides and appears to be important in decreasing their toxicity and carcinogenicity, although it is involved in the metabolic activation of certain carcinogens. Low dietary protein depresses epoxide hydratase activity in our dietary model (8). This may indicate both a decreased ability to detoxify epoxides and a decrease in metabolic activation of specific carcinogens, such as benzo(a)pyrene. Woodcock and Wood (19) have reported that dietary protein deficiency increases the activity of uridine diphosphate glucuronic acid (UDP6) transferase activity. This indicates that... 

Benson, A. M., Cha, Y.-N., Bueding, E., Heine, H. S., and Talalay, P., Elevation of extrahepatic glutathione S-transferase and epoxide hydratase activities by 2(3)-tert-butyl-4-hydroxyanisole. Cancer Res. 39, 2971-2977 (1979). 

The following values for the pharmacokinetic parameters were scaled up from vitro determinations of styrene monooxygenase and epoxide hydratase activities in mouse liver (15). 

Epoxide hydratase activity towards styrene-7,8-oxide, octene-1,2-oxide and benzo[a]pyrene-4,5-oxide has been detected variously in four species of bivalves (Table 12 Bendetal. 1977 Gallietal. 1988). Activity was similarin whole body and digestive gland microsomes of M. galloprovincialis the latter had a pH optimum... 

Another important group of hydrolytic enzymes are the epoxide hydrolases, also known as epoxide hydrase or epoxide hydratase, most commonly found in liver tissue. Epoxide hydrolases catalyze the hydration of arene oxides and aliphatic epoxides to their corresponding /raKt-dihydrodiols or diols, respectively, by activating a water molecule to attack one of the carbons of the arene oxide or epoxide [41]. Although one of the major metabolites of carbamazepine is the stable epoxide, this metabolite also undergoes hydrolysis to form the trans-dial metabolite (Fig. 3). Likewise, the anticonvulsants phenytoin and mephenytoin form arene oxides, which then form trans-dihy-drodiol that undergo further oxidation to form the catechols by the enzyme dihydrodiol dehydrogenase (Fig. 11) [42,43]. 

The most widely accepted pathway of PAH activation to yield DNA adducts involves the formation of anti- or syn-diol epoxides in the bay region [12, 13], Their formation is mediated by the sequential reaction of cytochrome P450 and epoxide hydratase, and is best described using the example of B[a]P. 

Epoxide hydratase is primarily microsomal (James and Little 1980) and present in highest activity in hepatopancreas (Table 20) the enzyme is also found in other tissues, viz. in nmol min mg microsomal protein, green gland 0.1 to 5.7 gill 0.4 to 3.5 egg masses 0.1 to 3.9 (James etal. 1979a see Table 20 for species and substrates). The activity was similar in hepatopancreas of male and female C. sapidus , and had a pH optimum of 6.6 to 12 (substrate BaP 4,5-oxide) and 7 to 7.8 (styrene oxide and octene oxide) H. americanus), and, for styrene oxide, a temperature optimum of 45 to 50 P. argus) or 35 to 40 (C. sapidus) and an... 

In addition to direct oxygenation, e.g. by aryl hydrocarbon hydroxylase, oxidative N- or 0-dealkylation is another process catalyzed by components of the Cytochrome P-450 System (mixed-function oxidases). Reduction also occurs in this system NADPH-cytochrome P-450 reductase has an activity similar to microsomal nitroreductase, i.e. transformation of aronaatic nitro compounds into the corresponding arylamines takes place. The oxidation may be followed by other enzymic reactions, e.g. epoxides are hydrated to vicinal diols by microsomal epoxide hydratase or they are coupled with glutathione by glutathione-S-epoxide transferase. 

To conclude, binding to subcellular structures and structure--activity relationship for the drugs as substrates of epoxide synthetase and of the epoxides as substrates or inhibitors of epoxide hydratase (46), using hepatic microsomal preparations, are also under study to try to give us a clearer understanding of the mechanisms of action, formation and inactivation of these intermediates. 

In summary, the formation of optically active compounds through hydrolysis reactions is dominated by biocatalysis mainly due to the availability and ease of use of a wide variety of esterases, lipases and (to a lesser extent) acylases. Epoxide ring-opening (and related reactions) is likely to be dominated by salen-metal catalysts while enzyme-catalysed nitrile hydrolysis seems destined to remain under-exploited until nitrilases or nitrile hydratases become commercially available. 

SP 361 SP 409 Immobilized enzyme mixture from Rhodococcus sp. containing nitrilase, nitril hydratase,esterase, epoxide hydrolase and amidase activity discontinued... 

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