Biochemical evidence for the involvement of tyrosine in epoxide activation during the catalytic cycle of epoxide hydrolase

Epoxide hydrolases (EH) catalyze the hydrolysis of epoxides and arene oxides to their corresponding diols. The crystal structure of murine soluble EH suggests that Tyr(465) and Tyr(381) act as acid catalysts, activating the epoxide ring and facilitating the formation of a covalent intermediate between the epoxide and the enzyme. To explore the role of these two residues, mutant enzymes were produced and the mechanism of action was analyzed. Enzyme assays on a series of substrates confirm that both Tyr(465) and Tyr(381) are required for full catalytic activity. The kinetics of chalcone oxide hydrolysis show that mutation of Tyr(465) and Tyr(381) decreases the rate of binding and the formation of an intermediate, suggesting that both tyrosines polarize the epoxide moiety to facilitate ring opening. These two tyrosines are, however, not implicated in the hydrolysis of the covalent intermediate. Sequence comparisons showed that Tyr(465) is conserved in microsomal EHs. The substitution of analogous Tyr(374) with phenylalanine in the human microsomal EH dramatically decreases the rate of hydrolysis of cis-stilbene oxide. These results suggest that these tyrosines perform a significant mechanistic role in the substrate activation by EHs.     

The molecular structure of epoxide hydrolase B from Mycobacterium tuberculosis and its complex with a urea-based inhibitor

Mycobacterium tuberculosis (Mtb), the intracellular pathogen that infects macrophages primarily, is the causative agent of the infectious disease tuberculosis in humans. The Mtb genome encodes at least six epoxide hydrolases (EHs A to F). EHs convert epoxides to trans-dihydrodiols and have roles in drug metabolism as well as in the processing of signaling molecules. Herein, we report the crystal structures of unbound Mtb EHB and Mtb EHB bound to a potent, low-nanomolar (IC₅₀ ≈ 19 nM) urea-based inhibitor at 2.1 and 2.4 Å resolution, respectively. The enzyme is a homodimer; each monomer adopts the classical α/β hydrolase fold that composes the catalytic domain; there is a cap domain that regulates access to the active site. The catalytic triad, comprising Asp104, His333 and Asp302, protrudes from the catalytic domain into the substrate binding cavity between the two domains. The urea portion of the inhibitor is bound in the catalytic cavity, mimicking, in part, the substrate binding; the two urea nitrogen atoms donate hydrogen bonds to the nucleophilic carboxylate of Asp104, and the carbonyl oxygen of the urea moiety receives hydrogen bonds from the phenolic oxygen atoms of Tyr164 and Tyr272. The phenolic oxygen groups of these two residues provide electrophilic assistance during the epoxide hydrolytic cleavage. Upon inhibitor binding, the binding-site residues undergo subtle structural rearrangement. In particular, the side chain of Ile137 exhibits a rotation of around 120° about its C^α-C^β bond in order to accommodate the inhibitor. These findings have not only shed light on the enzyme mechanism but also have opened a path for the development of potent inhibitors with good pharmacokinetic profiles against all Mtb EHs of the α/β type.     

Structure of human epoxide hydrolase reveals mechanistic inferences on bifunctional catalysis in epoxide and phosphate ester hydrolysis

The X-ray crystal structure of human soluble epoxide hydrolase (sEH) has been determined at 2.6 A resolution, revealing a domain-swapped quaternary structure identical to that observed for the murine enzyme [Argiriadi, M. A., Morisseau, C., Hammock, B. D., and Christianson, D. W. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 10637-10642]. As with the murine enzyme, the epoxide hydrolytic mechanism of the human enzyme proceeds through an alkyl-enzyme intermediate with Asp-333 in the C-terminal domain. The structure of the human sEH complex with N-cyclohexyl-N'-(iodophenyl)urea (CIU) has been determined at 2.35 A resolution. Tyr-381 and Tyr-465 donate hydrogen bonds to the alkylurea carbonyl group of CIU, consistent with the proposed roles of these residues as proton donors in the first step of catalysis. The N-terminal domain of mammalian sEH contains a 15 A deep cleft, but its biological function is unclear. Recent experiments demonstrate that the N-terminal domain of human sEH catalyzes the metal-dependent hydrolysis of phosphate esters [Cronin, A., Mowbray, S., Dürk, H., Homburg, S., Fleming, I., Fisslthaler, B., Oesch, F., and Arand, M. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 1552-1557; Newman, J. W., Morisseau, C., Harris, T. R., and Hammock, B. D. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 1558-1563]. The binding of Mg(2+)-HPO4(2-) to the N-terminal domain of human sEH in its CIU complex reveals structural features relevant to those of the enzyme-substrate complex in the phosphatase reaction.     

Polymorphisms in human soluble epoxide hydrolase: effects on enzyme activity, enzyme stability, and quaternary structure

Human soluble epoxide hydrolase (hsEH) has been shown to play a role in regulating blood pressure and inflammation. HsEH consists of an N-terminal phosphatase and a C-terminal epoxide hydrolase domain. In the present study, we examined the effects of polymorphisms in the hsEH gene on phosphatase activity, enzyme stability, and protein quaternary structure. The results showed that mutants Lys55Arg, Arg103Cys, Cys154Tyr, Arg287Gln, and the Arg103Cys/Arg287Gln (double mutant) have significantly lower phosphatase activity compared to the most frequent allele (MFA) of hsEH. In addition, the Lys55Arg, Arg103Cys, Cys154Tyr, Arg287Gln, and the double mutant have significantly lower kcat/Km values. The stabilities at 37 degrees C of purified Arg287Gln and Arg103Cys/Arg287Gln mutants were also significantly reduced compared to the MFA. HPLC size-exclusion studies showed that the MFA exists predominantly as a dimer. However, the Arg287Gln and Arg103Cys/Arg287Gln mutants show increased concentration of the monomer. We conclude that the Arg287Gln polymorphism disrupts putative intra- and inter-monomeric salt-bridges responsible for dimerization.     

Tyrosine residues serve as proton donor in the catalytic mechanism of epoxide hydrolase from Agrobacterium radiobacter

Epoxide hydrolase from Agrobacterium radiobacter catalyzes the hydrolysis of epoxides to their diols via an alkyl-enzyme intermediate. The recently solved X-ray structure of the enzyme shows that two tyrosine residues (Tyr152 and Tyr215) are positioned close to the nucleophile Asp107 in such a way that they can serve as proton donor in the alkylation reaction step. The role of these tyrosines, which are conserved in other epoxide hydrolases, was studied by site-directed mutagenesis. Mutation of Tyr215 to Phe and Ala and mutation of Tyr152 to Phe resulted in mutant enzymes of which the k(cat) values were only 2-4-fold lower than for wild-type enzyme, whereas the K(m) values for the (R)-enantiomers of styrene oxide and p-nitrostyrene oxide were 3 orders of magnitude higher than the K(m) values of wild-type enzyme, showing that the alkylation half-reaction is severely affected by the mutations. Pre-steady-state analysis of the conversion of (R)-styrene oxide by the Y215F and Y215A mutants showed that the 1000-fold elevated K(m) values were mainly caused by a 15-40-fold increase in K(S) and a 20-fold reduction in the rate of alkylation. The rates of hydrolysis of the alkyl-enzyme intermediates were not significantly affected by the mutations. The double mutant Y152F+Y215F showed only a low residual activity for (R)-styrene oxide, with a k(cat)/K(m) value that was 6 orders of magnitude lower than with wild-type enzyme and 3 orders of magnitude lower than with the single tyrosine mutants. This indicates that the effects of the mutations were cumulative. The side chain of Gln134 is positioned in the active site of the X-ray structure of epoxide hydrolase. Mutation of Gln134 to Ala resulted in an active enzyme with slightly altered steady-state kinetic parameters compared to wild-type enzyme, indicating that Gln134 is not essential for catalysis and that the side chain of Gln134 mimics bound substrate. Based upon this observation, the inhibitory potential of various unsubstituted amides was tested, resulting in the identification of phenylacetamide as a competitive inhibitor with an inhibition constant of 30 microM.     

Soybean epoxide hydrolase: identification of the catalytic residues and probing of the reaction mechanism with secondary kinetic isotope effects

Soybean epoxide hydrolase catalyzes the oxirane ring opening of 9,10-epoxystearate via a two-step mechanism involving the formation of an alkylenzyme intermediate, which, in contrast to most epoxide hydrolases studied so far, was found to be the rate-limiting step. We have probed residues potentially involved in catalysis by site-directed mutagenesis. Mutation of His(320), a residue predicted from sequence analysis to belong to the catalytic triad of the enzyme, considerably slowed down the second half-reaction. This kinetic manipulation provoked an accumulation of the reaction intermediate, which could be trapped and characterized by electrospray ionization mass spectrometry. As expected, mutation of Asp(126) totally abolished the activity of the enzyme from its crucial function as nucleophile involved in the formation of the alkylenzyme. In line with its role as the partner of His(320) in the "charge relay system," mutation of Asp(285) dramatically reduced the rate of catalysis. However, the mutant D285L still exhibited a very low residual activity, which, by structural analysis and mutagenesis, has been tentatively attributed to Glu(195), another acidic residue of the active site. Our studies have also confirmed the fundamental role of the conserved Tyr(175) and Tyr(255) residues, which are believed to activate the oxirane ring. Finally, we have determined the secondary tritium kinetic isotope effects on the epoxide opening step of 9,10-epoxystearate. The large observed values, i.e. (T)(V/K(m)) approximately 1.30, can be interpreted by the occurrence of a very late transition state in which the epoxide bond is broken before the nucleophilic attack by Asp(126) takes place.     

The x-ray structure of epoxide hydrolase from Agrobacterium radiobacter AD1. An enzyme to detoxify harmful epoxides.

Epoxide hydrolases catalyze the cofactor-independent hydrolysis of reactive and toxic epoxides. They play an essential role in the detoxification of various xenobiotics in higher organisms and in the bacterial degradation of several environmental pollutants. The first x-ray structure of one of these, from Agrobacterium radiobacter AD1, has been determined by isomorphous replacement at 2.1-A resolution. The enzyme shows a two-domain structure with the core having the alpha/beta hydrolase-fold topology. The catalytic residues, Asp107 and His275, are located in a predominantly hydrophobic environment between the two domains. A tunnel connects the back of the active-site cavity with the surface of the enzyme and provides access to the active site for the catalytic water molecule, which in the crystal structure, has been found at hydrogen bond distance to His275. Because of a crystallographic contact, the active site has become accessible for the Gln134 side chain, which occupies a position mimicking a bound substrate. The structure suggests Tyr152/Tyr215 as the residues involved in substrate binding, stabilization of the transition state, and possibly protonation of the epoxide oxygen.     

Localization of bound water in the solution structure of the immunoglobulin binding domain of streptococcal protein G. Evidence for solvent-induced helical distortion in solution.

The presence of bound water in the solution structure of the IgG binding domain of streptococcal protein G has been investigated by nuclear magnetic resonance using three-dimensional 1H rotating frame Overhauser 1H-15N multiple quantum coherence spectroscopy. The backbone amide protons of three residues, Ala20, Gln32 and Tyr33, are found to be in close proximity to bound water. Examination of the three-dimensional structure of the IgG binding domain indicates that in the vicinity of these three residues there are no backbone groups that do not already participate in hydrogen bonding and there are no suitably placed side-chain groups available for hydrogen bonding with water. As the lifetime of the bound water detected in this nuclear magnetic resonance experiment is greater than about one nanosecond, it is likely that the two bound water molecules participate in a bifurcating hydrogen bonding network comprising a CO-NH hydrogen bonded pair, such that the water molecule accepts a hydrogen bond from the NH proton and donates one to the carbonyl oxygen with the result that the amide proton is involved in a three center hydrogen bond. On the basis of the structure, one water molecule participates in such an interaction with the Ala20(NH)-Met1(CO) hydrogen bonded pair at the beginning of an anti-parallel beta-sheet, and the other with the Tyr33(NH)-Val29(CO) hydrogen bonded pair in the single alpha-helix. The latter, which is external and solvent accessible, is associated with a distortion in the alpha-helix centered around Tyr33 which consists of a significant increase in the CO(i-4)-N(i) and CO(i-4)-NH(i) distances relative to those in the rest of the helix, as well as a significant departure in the phi, psi angles of Tyr33 relative to regular helical geometry. Such solvent induced distortions in alpha-helices have been previously noticed in crystal structures and were postulated as possible folding intermediates for helical structures. The present observation of this phenomenon in solution indicates, however, that these water molecules are tightly bound and represent an integral part of the protein framework.     

The structure-activity relationship of urea derivatives as anti-tuberculosis agents

The treatment of tuberculosis is becoming more difficult due to the ever increasing prevalence of drug resistance. Thus, it is imperative that novel anti-tuberculosis agents, with unique mechanisms of action, be discovered and developed. The direct anti-tubercular testing of a small compound library led to discovery of adamantyl urea hit compound 1. In this study, the hit was followed up through the synthesis of an optimization library. This library was generated by systematically replacing each section of the molecule with a similar moiety until a clear structure-activity relationship was obtained with respect to anti-tubercular activity. The best compounds in this series contained a 1-adamantyl-3-phenyl urea core and had potent activity against Mycobacterium tuberculosis plus an acceptable therapeutic index. It was noted that the compounds identified and the pharmacophore developed is consistent with inhibitors of epoxide hydrolase family of enzymes. Consequently, the compounds were tested for inhibition of representative epoxide hydrolases: M. tuberculosis EphB and EphE; and human soluble epoxide hydrolase. Many of the optimized inhibitors showed both potent EphB and EphE inhibition suggesting the antitubercular activity is through inhibition of multiple epoxide hydrolase enzymes. The inhibitors also showed potent inhibition of humans soluble epoxide hydrolase, but limited cytotoxicity suggesting that future studies must be towards increasing the selectivity of epoxide hydrolase inhibition towards the M. tuberculosis enzymes.     

Human soluble epoxide hydrolase: structural basis of inhibition by 4-(3-cyclohexylureido)-carboxylic acids

X-ray crystal structures of human soluble epoxide hydrolase (sEH) complexed with four different dialkylurea inhibitors bearing pendant carboxylate "tails" of varying length have been determined at 2.3-3.0 A resolution. Similarities among inhibitor binding modes reinforce the proposed roles of Y381 and/or Y465 as general acids that protonate the epoxide ring of the substrate in concert with nucleophilic attack of D333 at the electrophilic epoxide carbon. Additionally, the binding of these inhibitors allows us to model the binding mode of the endogenous substrate 14,15-epoxyeicosatrienoic acid. Contrasts among inhibitor binding modes include opposite orientations of inhibitor binding in the active-site hydrophobic tunnel. Alternative binding orientations observed for this series of inhibitors to human sEH, as well as the binding of certain dialkylurea inhibitors to human sEH and murine sEH, complicate the structure-based design of human sEH inhibitors with potential pharmaceutical applications in the treatment of hypertension. Thus, with regard to the optimization of inhibitor designs targeting human sEH, it is critical that human sEH and not murine sEH be utilized for inhibitor screening, and it is critical that structures of human sEH-inhibitor complexes be determined to verify inhibitor binding orientations that correlate with measured affinities.     

Stabilization of the transition state for the transfer of tyrosine to tRNA(Tyr) by tyrosyl-tRNA synthetase

Aminoacylation of tRNA(Tyr) involves two steps: (1) tyrosine activation to form the tyrosyl-adenylate intermediate; and (2) transfer of tyrosine from the tyrosyl-adenylate intermediate to tRNA(Tyr). In Bacillus stearothermophilus tyrosyl-tRNA synthetase, Asp78, Tyr169, and Gln173 have been shown to form hydrogen bonds with the alpha-ammonium group of the tyrosine substrate during the first step of the aminoacylation reaction. Asp194 and Gln195 stabilize the transition state complex for the first step of the reaction by hydrogen bonding with the 2'-hydroxyl group of AMP and the carboxylate oxygen atom of tyrosine, respectively. Here, the roles that Asp78, Tyr169, Gln173, Asp194, and Gln195 play in catalysis of the second step of the reaction are investigated. Pre-steady-state kinetic analyses of alanine variants at each of these positions shows that while the replacement of Gln173 by alanine does not affect the initial binding of the tRNA(Tyr) substrate, it destabilizes the transition state complex for the second step of the reaction by 2.3 kcal/mol. None of the other alanine substitutions affects either the initial binding of the tRNA(Tyr) substrate or the stability of the transition state for the second step of the aminoacylation reaction. Taken together, the results presented here and the accompanying paper are consistent with a concerted reaction mechanism for the transfer of tyrosine to tRNA(Tyr), and suggest that catalysis of the second step of tRNA(Tyr) aminoacylation involves stabilization of a transition state in which the scissile acylphosphate bond of the tyrosyl-adenylate species is strained. Cleavage of the scissile bond on the breakdown of the transition state alleviates this strain.     

QM/MM study of the mechanism of enzymatic limonene 1,2-epoxide hydrolysis

Limonene 1,2-epoxide hydrolase (LEH) is completely different from those of classic epoxide hydrolases (EHs) which catalyze the hydrolysis of epoxides to vicinal diols. A novel concerted general acid catalysis step involving the Asp101-Arg99-Asp132 triad is proposed to play an important role in the mechanism. Combined quantum-mechanical/molecular-mechanical (QM/MM) calculations gave activation barriers of 16.9 and 25.1 kcal/mol at the B3LYP/6-31G(d,p)//CHARMM level for nucleophilic attack on the more and less substituted epoxide carbons, respectively. Furthermore, the important roles of residues Arg99, Tyr53 and Asn55 on mutated LEH were evaluated by QM/MM-scanned energy mapping. These results may provide an explanation for site-directed mutagenesis.     

Implications for an ionized alkyl-enzyme intermediate during StEH1-catalyzed trans-stilbene oxide hydrolysis

The catalytic mechanism of epoxide hydrolase (EC 3.3.2.3) involves acid-assisted ring opening of the oxirane during the alkylation half-reaction of hydrolysis. Two tyrosyl residues in the active site of epoxide hydrolases have been shown to contribute to the catalysis of enzyme alkylation, but their mechanism of action has not been fully described. We have investigated the involvement of the active site Tyr154 and Tyr235 during S,S-trans-stilbene oxide hydrolysis catalyzed by potato epoxide hydrolase StEH1. Tyr phenol ionizations of unliganded enzyme as well as under pre-steady-state conditions during catalysis were studied by direct absorption spectroscopy. A transient UV absorption, indicative of tyrosinate formation, was detected during the lifetime of the alkyl-enzyme intermediate. The apparent pKa of Tyr ionization was 7.3, a value more than 3 pH units below the estimated pKa of protein Tyr residues in the unliganded enzyme. In addition, the pH dependencies of microscopic kinetic rates of catalyzed S,S-trans-stilbene oxide hydrolysis were determined. The alkylation rate increased with pH and displayed a pKa value identical to that of Tyr ionization (7.3), whereas the reverse (epoxidation) reaction did not display any pH dependence. The rate of alkyl-enzyme hydrolysis was inversely dependent on tyrosinate formation, decreasing with its buildup in the active site. Since alkyl-enzyme hydrolysis is the rate-limiting step of the overall reaction, kcat displayed the same decrease with pH as the hydrolysis rate. The compiled results suggested that the role of the Tyr154/Tyr235 pair was not as ultimate proton donor to the alkoxide anion but to stabilize the negatively charged alkyl-enzyme through electrophilic catalysis via hydrogen bonding.     

Expression and characterization of an epoxide hydrolase from Anopheles gambiae with high activity on epoxy fatty acids

In insects, epoxide hydrolases (EHs) play critical roles in the metabolism of xenobiotic epoxides from the food resources and in the regulation of endogenous chemical mediators, such as juvenile hormones. Using the baculovirus expression system, we expressed and characterized an epoxide hydrolase from Anopheles gambiae (AgEH) that is distinct in evolutionary history from insect juvenile hormone epoxide hydrolases (JHEHs). We partially purified the enzyme by ion exchange chromatography and isoelectric focusing. The experimentally determined molecular weight and pI were estimated to be 35 kD and 6.3 respectively, different than the theoretical ones. The AgEH had the greatest activity on long chain epoxy fatty acids such as 14,15-epoxyeicosatrienoic acids (14,15-EET) and 9,10-epoxy-12Z-octadecenoic acids (9,10-EpOME or leukotoxin) among the substrates evaluated. Juvenile hormone III, a terpenoid insect growth regulator, was the next best substrate tested. The AgEH showed kinetics comparable to the mammalian soluble epoxide hydrolases, and the activity could be inhibited by AUDA [12-(3-adamantan-1-yl-ureido) dodecanoic acid], a urea-based inhibitor designed to inhibit the mammalian soluble epoxide hydrolases. The rabbit serum generated against the soluble epoxide hydrolase of Mus musculus can both cross-react with natural and denatured forms of the AgEH, suggesting immunologically they are similar. The study suggests there are mammalian sEH homologs in insects, and epoxy fatty acids may be important chemical mediators in insects.     

EH3 (ABHD9): the first member of a new epoxide hydrolase family with high activity for fatty acid epoxides

Epoxide hydrolases are a small superfamily of enzymes important for the detoxification of chemically reactive xenobiotic epoxides and for the processing of endogenous epoxides that act as signaling molecules. Here, we report the identification of two human epoxide hydrolases: EH3 and EH4. They share 45% sequence identity, thus representing a new family of mammalian epoxide hydrolases. Quantitative RT-PCR from mouse tissue indicates strongest EH3 expression in lung, skin, and upper gastrointestinal tract. The recombinant enzyme shows a high turnover number with 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid (EET), as well as 9,10-epoxyoctadec-11-enoic acid (leukotoxin). It is inhibited by a subclass of N,N'-disubstituted urea derivatives, including 12-(3-adamantan-1-yl-ureido)-dodecanoic acid, 1-cyclohexyl-3-dodecylurea, and 1-(1-acetylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea, compounds so far believed to be selective inhibitors of mammalian soluble epoxide hydrolase (sEH). Its sensitivity to this subset of sEH inhibitors may have implications on the pharmacologic profile of these compounds. This is particularly relevant because sEH is a potential drug target, and clinical trials are under way exploring the value of sEH inhibitors in the treatment of hypertension and diabetes type II.     

Homology modeling of a novel epoxide hydrolase (EH) from Aspergillus niger SQ-6: structure-activity relationship in expoxides inhibiting EH activity

The 3D structure of a novel epoxide hydrolase from Aspergillus niger SQ-6 (sqEH) was constructed by using homology modeling and molecular dynamics simulations. Based on the 3D model, Asp191, His369 and Glu343 were predicted as catalytic triad. The putative active pocket is a hydrophobic environment and is rich in some important non—polar residues (Pro318, Trp282, Pro319, Pro317 and Phe242). Using three sets of epoxide inhibitors for docking study, the interaction energies of sqEH with each inhibitor are consistent with their inhibitory effects in previous experiments. Moreover, a critical water molecule which closes to the His369 was identified to be an ideal position for the hydrolysis step of the reaction. Two tyrosine residues (Tyr249 and Tyr312) are able to form hydrogen bonds with the epoxide oxygen atom to maintain the initial binding and positioning of the substrate in the active pocket. These docked complex models can well interpret the substrate specificity of sqEH, which could be relevant for the structural—based design of specific epoxide inhibitors. Figure       

Epoxide hydrolases: their roles and interactions with lipid metabolism

The epoxide hydrolases (EHs) are enzymes present in all living organisms, which transform epoxide containing lipids by the addition of water. In plants and animals, many of these lipid substrates have potent biologically activities, such as host defenses, control of development, regulation of inflammation and blood pressure. Thus the EHs have important and diverse biological roles with profound effects on the physiological state of the host organisms. Currently, seven distinct epoxide hydrolase sub-types are recognized in higher organisms. These include the plant soluble EHs, the mammalian soluble epoxide hydrolase, the hepoxilin hydrolase, leukotriene A4 hydrolase, the microsomal epoxide hydrolase, and the insect juvenile hormone epoxide hydrolase. While our understanding of these enzymes has progressed at different rates, here we discuss the current state of knowledge for each of these enzymes, along with a distillation of our current understanding of their endogenous roles. By reviewing the entire enzyme class together, both commonalities and discrepancies in our understanding are highlighted and important directions for future research pertaining to these enzymes are indicated.     

Exploration of secondary and tertiary pharmacophores in unsymmetrical N,N′-diaryl urea inhibitors of soluble epoxide hydrolase

The impact of various secondary and tertiary pharmacophores on in vitro potency of soluble epoxide hydrolase (sEH) inhibitors based on the unsymmetrical urea scaffold 1 is discussed. N,N′-Diaryl urea inhibitors of soluble epoxide hydrolase exhibit subtle variations in inhibitory potency depending on the secondary pharmacophore but tolerate considerable structural variation in the second linker/tertiary pharmacophore fragment.     

Physico-chemical properties of the epoxide hydrolase from Rhodosporidium toruloides

Residue-specific chemical modification of amino acid residues of the microsomal epoxide hydrolase (mEH) from Rhodosporidium toruloides UOFS Y-0471 revealed that the enzyme is inactivated through modification of Asp/Glu and His residues, as well as through modification of Ser. Since Asp acts as the nucleophile, and Asp/Glu and His serve as charge relay partners in the catalytic triad of microsomal and soluble epoxide hydrolases during epoxide hydrolysis, inactivation of the enzyme by modification of the Asp/Glu and His residues agrees with the established reaction mechanism of these enzymes. However, the inactivation of the enzyme through modification of Ser residues is unexpected, suggesting that a Ser in the catalytic site is indispensable for substrate binding by analogy of the role of Ser residues in the related L-2-haloacid dehalogenases, as well as the ATPase and phosphatase enzymes. Co²⁺, Hg²⁺, Ag⁺, Mg²⁺ and Ca²⁺ inhibited enzyme activity and EDTA increased enzyme activity. The activation energy for inactivation of the enzyme was 167 kJ mol^–1. Kinetic constants for the enzyme could not be determined since unusual behaviour was displayed during hydrolysis of 1,2-epoxyoctane by the purified enzyme. Enantioselectivity w as strongly dependent on substrate concentration. When the substrate was added in concentrations ensuring two-phase conditions, the enantioselectivity was greatly enhanced. On the basis of these results, it is proposed that this enzyme acts at an interface, analogous to lipases.     

Epoxide hydrolases: biochemistry and molecular biology

Epoxides are organic three-membered oxygen compounds that arise from oxidative metabolism of endogenous, as well as xenobiotic compounds via chemical and enzymatic oxidation processes, including the cytochrome P450 monooxygenase system. The resultant epoxides are typically unstable in aqueous environments and chemically reactive. In the case of xenobiotics and certain endogenous substances, epoxide intermediates have been implicated as ultimate mutagenic and carcinogenic initiators Adams et al. (Chem. Biol. Interact. 95 (1995) 57-77) Guengrich (Properties and Metabolic roles 4 (1982) 5-30) Sayer et al. (J. Biol. Chem. 260 (1985) 1630-1640). Therefore, it is of vital importance for the biological organism to regulate levels of these reactive species. The epoxide hydrolases (E.C. 3.3.2. 3) belong to a sub-category of a broad group of hydrolytic enzymes that include esterases, proteases, dehalogenases, and lipases Beetham et al. (DNA Cell Biol. 14 (1995) 61-71). In particular, the epoxide hydrolases are a class of proteins that catalyze the hydration of chemically reactive epoxides to their corresponding dihydrodiol products. Simple epoxides are hydrated to their corresponding vicinal dihydrodiols, and arene oxides to trans-dihydrodiols. In general, this hydration leads to more stable and less reactive intermediates, however exceptions do exist. In mammalian species, there are at least five epoxide hydrolase forms, microsomal cholesterol 5,6-oxide hydrolase, hepoxilin A(3) hydrolase, leukotriene A(4) hydrolase, soluble, and microsomal epoxide hydrolase. Each of these enzymes is distinct chemically and immunologically. Table 1 illustrates some general properties for each of these classes of hydrolases. Fig. 1 provides an overview of selected model substrates for each class of epoxide hydrolase.