The Catalytic Mechanism of Microsomal Epoxide Hydrolase Involves Reversible Formation and Rate-Limiting Hydrolysis of the Alkyl−Enzyme Intermediate

Tzeng, Huey-Fen; Laughlin, L T; Lin, and Sue; Armstrong, Richard N.

doi:10.1021/ja961826x

Cited by 65 publications

(63 citation statements)

References 12 publications

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“…Based on the established mechanism of EH-catalyzed hydration (Lacourciere and Armstrong, 1993;Tzeng, 1996;Laughlin et al, 1998;Armstrong, 1999), a modified pathway for the mEH-catalyzed hydration of the oxetanyl moiety in AZD1979 is proposed (Fig. 9).…”

Section: Discussionmentioning

confidence: 99%

Discovery of a Novel Microsomal Epoxide Hydrolase-Catalyzed Hydration of a Spiro Oxetane

Hayes

Grönberg

et al. 2016

Drug Metabolism and Disposition

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Oxetane moieties are increasingly being used by the pharmaceutical industry as building blocks in drug candidates because of their pronounced ability to improve physicochemical parameters and metabolic stability of drug candidates. The enzymes that catalyze the biotransformation of the oxetane moiety are, however, not well studied. The in vitro metabolism of a spiro oxetane-containing compound AZD1979 [(3-(4-(2-oxa-6-azaspiro[3.3]heptan-6-ylmethyl)phenoxy)azetidin-1-yl)(5-(4-ethoxyphenyl)-1,3,4-oxadiazol-2-yl)methanone] was studied and one of its metabolites, M1, attracted our interest because its formation was NAD(P)H independent. The focus of this work was to elucidate the structure of M1 and to understand the mechanism(s) of its formation. We established that M1 was formed via hydration and ring opening of the oxetanyl moiety of AZD1979. Incubations of AZD1979 using various human liver subcellular fractions revealed that the hydration reaction leading to M1 occurred mainly in the microsomal fraction. The underlying mechanism as a hydration, rather than an oxidation reaction, was supported by the incorporation of 18 O from H 2 18O into M1. Enzyme kinetics were performed probing the formation of M1 in human liver microsomes. The formation of M1 was substantially inhibited by progabide, a microsomal epoxide hydrolase inhibitor, but not by trans-4-[4-(1-adamantylcarbamoylamino)cyclohexyloxy]-benzoic acid, a soluble epoxide hydrolase inhibitor. On the basis of these results, we propose that microsomal epoxide hydrolase catalyzes the formation of M1. The substrate specificity of microsomal epoxide hydrolase should therefore be expanded to include not only epoxides but also the oxetanyl ring system present in AZD1979.

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Section: Discussionmentioning

confidence: 99%

Discovery of a Novel Microsomal Epoxide Hydrolase-Catalyzed Hydration of a Spiro Oxetane

Hayes

Grönberg

et al. 2016

Drug Metabolism and Disposition

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“…The alkylation rates displayed by A. radiobacter epoxide hydrolase with styrene oxide and microsomal epoxide hydrolase with glycidyl-4-nitrobenzoate were higher (10-100-fold) than the rate of TSO alkylation of StEH1-5H [33,34]. Simultaneously, the dissociation constants (K S ) of the two first enzymes were elevated at comparable magnitudes when compared with the K S TSO for StEH1-5H.…”

Section: Substrate Specificitymentioning

confidence: 91%

“…These steps, alkylation (ES → ES ) and hydrolysis (E → EP), have been demonstrated to be primarily responsible for the catalytic efficiency of rat microsomal epoxide hydrolase with glycidyl-4-nitrobenzoate as substrate and the A. radiobacter soluble epoxide hydrolase with styrene oxide as substrate. The alkylation step, with rate constant k 2 , was shown to be considerably slower than ES complex formation (k 1 ) but much faster than the rate of hydrolysis (k 3 ) [33,34]. Hence, the hydrolytic step is primarily setting the upper limit to the value of k cat in these enzymes.…”

Section: Substrate Specificitymentioning

confidence: 99%

Catalysis of potato epoxide hydrolase, StEH1

Elfström

Widersten

2005

Biochemical Journal

134

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The kinetic mechanism of epoxide hydrolase (EC 3.3.2.3) from potato, StEH1 (Solanum tuberosum epoxide hydrolase 1), was studied by presteady-state and steady-state kinetics as well as by pH dependence of activity. The specific activities towards the different enantiomers of TSO (trans-stilbene oxide) as substrate were 43 and 3 µmol · min −1 · mg −1 with the R,R-or S,S-isomers respectively. The enzyme was, however, enantioselective in favour of the S,S enantiomer due to a lower K m value. The pH dependences of k cat with R,R or S,S-TSO were also distinct and supposedly reflecting the pH dependences of the individual kinetic rates during substrate conversion. The rate-limiting step for TSO and cis-and trans-epoxystearate was shown by rapid kinetic measurements to be the hydrolysis of the alkylenzyme intermediate. Functional characterization of point mutants verified residues Asp 105 , Tyr 154 , Tyr 235 and His 300 as crucial for catalytic activity. All mutants displayed drastically decreased enzymatic activities during steady state. Presteady-state measurements revealed the base-deficient H300N (His 300 → Asn) mutant to possess greatly reduced efficiencies in catalysis of both chemical steps (alkylation and hydrolysis).

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“…In the case of enzymatic epoxide hydrolysis, the first step of the reaction proceeds significantly faster than the second step, which therefore becomes rate-limiting. Armstrong (1999) calculated, for glycidyl-4-nitrobenzoate as the substrate, the rate constant of step 1 to be three orders of magnitude higher than the rate constant for step 2 (Tzeng et al, 1996). According to our own work, similar reaction kinetics exist for the turnover of 9,10-epoxystearic acid (Arand et al, 1999) and styrene oxide.…”

Section: Fast Detoxification By the Microsomal Epoxide Hydrolase Despmentioning

confidence: 62%

“…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).…”

Section: Fast Detoxification By the Microsomal Epoxide Hydrolase Despmentioning

confidence: 99%

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

Oesch

Hengstler

Arand

2004

Nonlinearity in Biology, Toxicology, Medicine

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The Catalytic Mechanism of Microsomal Epoxide Hydrolase Involves Reversible Formation and Rate-Limiting Hydrolysis of the Alkyl−Enzyme Intermediate

Cited by 65 publications

References 12 publications

Discovery of a Novel Microsomal Epoxide Hydrolase-Catalyzed Hydration of a Spiro Oxetane

Discovery of a Novel Microsomal Epoxide Hydrolase-Catalyzed Hydration of a Spiro Oxetane

Catalysis of potato epoxide hydrolase, StEH1

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

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