Visualization of a Covalent Intermediate between Microsomal Epoxide Hydrolase, but not Cholesterol Epoxide Hydrolase, and their Substrates

Abstract

Mammalian soluble and microsomal epoxide hydrolases have been proposed to belong to the family of n/~-hydrolase-fold enzymes. These enzymes hydrolyse their substrates by a catalytic triad, with the first step of the enzymatic reaction being the formation of a covalent enzyme-substrate ester. In the present paper, we describe the direct visualization of the ester formation between rat microsomal epoxide hydrolase and its substrate. Microsomal epoxide hydrolase was precipitated with acetone after brief incubation with 11 -"C]epoxystearic acid. After denaturing SDS gel electrophoresis the protein-bound radioactivity was detected by fluorography. Pure epoxide hydrolase and crude microsomes showed a single radioactive signal of the expected molecular mass that could be suppressed by inclusion of the competitive inhibitor 1 ,I ,I -trichloropropene oxide in the incubation mixture. In a similar manner, 4-fluorochalconeoxide-senaitive binding of epoxystearic acid to rat soluble epoxide hydrolase could be demonstrated in rat liver cytosol. Under similar conditions, no covalent binding of [26-'JC]cholesterol-5a,6n-epoxide to microsomal proteins or solubilized fractions tenfold enriched in cholesterol epoxide hydrolase activity could be observed. Our data provide definitive proof for the formation of an enzyme-substrate-ester intermediate formed in the course of epoxide hydrolysis by microsomal epoxide hydrolase, show no formation of a covalent intermediate between cholesterol epoxide hydrolase and its substrate under the same conditions as those under which an intermediate was shown for both microsomal and soluble epoxide hydrolases and therefore indicate that the cholesterol epoxide hydrolase apparently does not act by a similar mechanism and is probably not structurally related to microsomal and soluble epoxide hydrolases.Keywords ; epoxide hydrolase ; mechanism ; a/b hydrolase fold ; cholesterol ; fatty acid metabolism.Epoxide hydrolases (EH) represent a group of ubiquitous enzymes with important functions in the detoxification of reactive intermediates, namely epoxides, that arise from a large variety of compounds during their metabolism. The two mammalian enzymes implicated in the metabolism of foreign compounds are microsomal EH (Oesch, 1973) and soluble EH (Ota and Hammock, 1980). Both enzymes have been cloned from a variety of species (Beetham et al., 1993;Grant et al., 1993;Jackson et al., 1987;Knehr et al., 1993;Porter et al., 1986;Wojtasek and Prestwich, 1996). A third enzyme, cholesterol EH, which is membrane bound similarly to inicrosomal EH but otherwise distinct from the latter (Oesch et al., 1984), is less well investigated. Its physiological function appears to be the conversion of 5n,6n-epoxycholestane-3P-o1 and SP,6D-epoxycholestane-3/!-ol, the two epoxides arising from cholesterol during, e.g. lipid peroxidation, to give the single product cholestane-3~,Scr,b/j-triol (Watabe et al., 1981).For a long time it was believed that EH convert their substrates by direct hydrolysis. The pioneering work of Hanzli...

Epoxide hydrolases: biochemistry and molecular biology

“…As indicated previously, cholesterol oxide hydrolase is a distinct enzyme from oxidosqualene cyclase, as well as from microsomal epoxide hydrolase, the latter being responsible for the metabolism of a wide range of xenobiotic alkene and arene oxides [4,1,5]. Although the cholesterol 5,6 b-oxide is more reactive than the a-oxide upon acid-catalyzed hydration, the a-oxide is a 4.5-fold better substrate than the b-oxide as indicated by values of V max /K m [1].…”

“…Unlike xenobiotic microsomal epoxide hydrolase, which is not inactivated or inhibited by 7-dehydrocholesterol 5,6 b-oxide, cholesterol oxide hydrolase appears to hydrolyze cholesterol oxides via a positively charged transition state [9]. The mammalian soluble and microsomal epoxide hydrolases have been proposed to belong to the family of a/b-hydrolase-fold enzymes, enzymes that hydrolyze their substrates by a catalytic triad, with the first step of the enzymatic reaction being the formation of a covalent enzyme -substrate ester [5]. Although an enzyme-substrate -ester intermediate is detected in the course of epoxide hydrolysis by microsomal and soluble epoxide hydrolases, no such covalent intermediate between cholesterol epoxide hydrolase and its substrate is detected, indicating that the cholesterol epoxide hydrolase does not, apparently, act by a similar mechanism and is probably not related structurally to microsomal and soluble epoxide hydrolases [5].…”

Biocatalytic conversion of epoxides

“…The expected vicinal diol is not formed owing to the nature of the substrate, which allows a shift of the carbocation intermediate. Other probable candidates for non-covalent catalysis are limonene epoxide hydrolase from Rhodococcus erythropolis [32] (see also Update) and cholesterol epoxide hydrolase [33]. It is conceivable that with such different enzymes alternative nucleophiles might be used.…”

Detoxication Strategy of Epoxide Hydrolase—The Basis for a Novel Threshold for Definable Genotoxic Carcinogens

“…However, even this is not sufficient to account for its role as a rapid detoxifier. Recent findings have led to a detailed understanding of the enzymatic mechanism by which mEH and the related soluble epoxide hydrolase hydrolyze their substrates Armstrong, 1993, 1994;Arand et al, 1994Arand et al, , 1996Hammock et al, 1994;Tzeng et al, 1996Tzeng et al, , 1998Müller et al, 1997;Laughlin et al, 1998;Arand et al, in press). These enzymes belong to the large structural family of α/β hydrolase fold enzymes (Ollis et al, 1992).…”