Title: Biochemical Evidence for the Involvement of Tyrosine in Epoxide Activation during the Catalytic Cycle of Epoxide Hydrolase
Abstract: Epoxide hydrolases (EH) catalyze the hydrolysis of epoxides and arene oxides to their corresponding diols. The crystal structure of murine soluble EH suggests that Tyr465and Tyr381 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 Tyr465 and Tyr381 are required for full catalytic activity. The kinetics of chalcone oxide hydrolysis show that mutation of Tyr465 and Tyr381 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 Tyr465 is conserved in microsomal EHs. The substitution of analogous Tyr374 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. Epoxide hydrolases (EH) catalyze the hydrolysis of epoxides and arene oxides to their corresponding diols. The crystal structure of murine soluble EH suggests that Tyr465and Tyr381 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 Tyr465 and Tyr381 are required for full catalytic activity. The kinetics of chalcone oxide hydrolysis show that mutation of Tyr465 and Tyr381 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 Tyr465 is conserved in microsomal EHs. The substitution of analogous Tyr374 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. epoxide hydrolase soluble epoxide hydrolase microsomal epoxide hydrolase 4-nitrophenyl-2,3-epoxy-3-phenylpropyl carbonate trans-1,3-diphenylpropene oxide cis-9,10-epoxystearic acid trans-stilbene oxide cis-stilbene oxide juvenile hormone III 4-fluorochalcone oxide Epoxide hydrolases (EH,1EC3.3.2.3) hydrolyze epoxides and arene oxides to their corresponding diols (1Oesch F. Xenobiotica. 1973; 3: 305-340Crossref PubMed Scopus (729) Google Scholar). These enzymes are widely distributed among many species, including bacteria, fungi, plants, insects, and mammals (2Rink R. Fennema M. Smids M. Dehmel U. Janssen D. J. Biol. Chem. 1997; 272: 14650-14657Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 3Arand M. Hemmer H. Durk H. Baratti J. Archelas A. Furstoss R. Oesch F. Biochem. J. 1999; 344: 273-280Crossref PubMed Scopus (89) Google Scholar, 4Debernard S. Morisseau C. Severson T.F. Feng L. Wojtasek H. Prestwich G.D. Hammock B.D. Insect Biochem. Mol. Biol. 1998; 28: 409-419Crossref PubMed Scopus (39) Google Scholar, 5Beetham J.K. Grant D. Arand M. Garbarino J. Kiyosue T. Pinot F. Oesch F. Belknap W.R. Shinozaki K. Hammock B.D. DNA Cell Biol. 1995; 14: 61-71Crossref PubMed Scopus (123) Google Scholar). In mammals, there are two major classes of EH with broad and complementary substrate selectivity, soluble EH (sEH) and microsomal EH (mEH) (6Hammock B. Grant D. Storms D. Sipes I. McQueen C. Gandolfi A. Comprehensive Toxicology. 3. Pergamon, Oxford1997: 283-305Google Scholar). sEH participates not only in xenobiotic detoxification but also endogenous lipid metabolism, acting on epoxides of linoleic acid (leukotoxin and isoleukotoxin) (7Moghaddam M.F. Grant D.F. Cheek J.M. Greene J.F. Williamson K.C. Hammmock B.D. Nat. Med. 1997; 3: 562-566Crossref PubMed Scopus (256) Google Scholar) and arachidonic acid (cis-epoxyeicosatrienoic acids) (8Zeldin D.C. Wei S. Falck J.R. Hammock B.D. Snapper J.R. Capdevila J.H. Arch. Biochem. Biophys. 1995; 316: 443-451Crossref PubMed Scopus (119) Google Scholar). Elevated titers of linoleate and arachidonic acid diols are, respectively, thought to be associated with the inflammatory disorder known as acute respiratory distress syndrome (7Moghaddam M.F. Grant D.F. Cheek J.M. Greene J.F. Williamson K.C. Hammmock B.D. Nat. Med. 1997; 3: 562-566Crossref PubMed Scopus (256) Google Scholar) and pregnancy-induced hypertension (9Oltman C. Weintraub N. VanRollins M. Dellsperger K. Circ. Res. 1998; 83: 932-939Crossref PubMed Scopus (213) Google Scholar). Inhibition of epoxide hydration may accordingly have a therapeutic value for these two serious disorders. Alternately, mEH appears to be mainly involved in the metabolism of xenobiotic epoxides (6Hammock B. Grant D. Storms D. Sipes I. McQueen C. Gandolfi A. Comprehensive Toxicology. 3. Pergamon, Oxford1997: 283-305Google Scholar). A protein-reactive and cytotoxic epoxide, naphthalene epoxide for example, is converted to the less toxic diol by this enzyme (10Tingle M. Pirmohamed M. Templeton E. Wilson A. Madden S. Kitteringham N. Park B. Biochem. Pharmacol. 1993; 46: 1529-1538Crossref PubMed Scopus (74) Google Scholar). The mEH is also related to activation of other arene oxides such as 7,12-dimethylbenzanthracene, a member of the polycyclic aromatic hydrocarbon class of chemical carcinogens (11Phillips D. Grover P. Drug. Metab. Rev. 1994; 26: 443-467Crossref PubMed Scopus (98) Google Scholar). To understand xenobiotic toxicity, metabolic aberrations associated with pathological disorders, and to develop possible therapies against these, it is important to elucidate the molecular basis of EH catalysis. The EHs belong to the α/β hydrolase fold family. These enzymes characteristically employ a two-step mechanism in which a catalytic nucleophile of the enzymes attacks a polarized electrophilic substrate, and the covalent intermediate is subsequently hydrolyzed (Fig. 1) (6Hammock B. Grant D. Storms D. Sipes I. McQueen C. Gandolfi A. Comprehensive Toxicology. 3. Pergamon, Oxford1997: 283-305Google Scholar). The mechanism of murine sEH has been mainly elucidated from a series of experiments utilizing heavy isotopes, protein mass spectrometry, and site-directed mutagenesis (12Pinot F. Grant D.F. Beetham J.K. Parker A.G. Borhan B. Landt S. Jones A.D. Hammock B.D. J. Biol. Chem. 1995; 270: 7968-7974Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 13Borhan B. Jones A.D. Pinot F. Grant D.F. Kurth M.J. Hammock B.D. J. Biol. Chem. 1995; 270: 26923-26930Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). They indicated that Asp333 acts as a catalytic nucleophile and that a water molecule is activated by the nearby His523 and Asp495 pair (Fig. 1). This mechanism was extended to other EHs (2Rink R. Fennema M. Smids M. Dehmel U. Janssen D. J. Biol. Chem. 1997; 272: 14650-14657Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 14Arand M. Wagner H. Oesch F. J. Biol. Chem. 1996; 271: 4223-4229Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 15Laughlin L.T. Tzeng H.F. Lin S. Armstrong R.N. Biochemistry. 1998; 37: 2897-2904Crossref PubMed Scopus (51) Google Scholar, 16Tzeng H.F. Laughlin L.T. Armstrong R.N. Biochemistry. 1998; 37: 2905-2911Crossref PubMed Scopus (46) Google Scholar, 17Arand M. Muller F. Mecky A. Hinz W. Urban P. Pompon D. Kellner R. Oesch F. Biochem. J. 1999; 337: 37-43Crossref PubMed Scopus (66) Google Scholar, 18Lacourciere G.M. Armstrong R.N. J. Am. Chem. Soc. 1993; 115: 10466-10467Crossref Scopus (153) Google Scholar). However, one or more additional amino acids are likely involved in the catalytic cycle especially in the activation of the epoxide ring (5Beetham J.K. Grant D. Arand M. Garbarino J. Kiyosue T. Pinot F. Oesch F. Belknap W.R. Shinozaki K. Hammock B.D. DNA Cell Biol. 1995; 14: 61-71Crossref PubMed Scopus (123) Google Scholar), based on the mechanism of the haloalkane dehalogenase, HLD1, from Xantobacter autotrophicus G10, a related α/β hydrolase (6Hammock B. Grant D. Storms D. Sipes I. McQueen C. Gandolfi A. Comprehensive Toxicology. 3. Pergamon, Oxford1997: 283-305Google Scholar). Kinetics of the hydrolysis of chalcone oxide by sEH support this hypothesis. Thus a generalized scheme is postulated in which one or more amino acid(s) may polarize the epoxide oxygen by an acid-like mechanism, weakening the C-O epoxide bond and facilitating the attack on the carbon of the epoxide ring by a nucleophile, such as the conjugate base, Asp333 (19Morisseau C. Du G. Newman J.W. Hammock B.D. Arch. Biochem. Biophys. 1998; 356: 214-228Crossref PubMed Scopus (73) Google Scholar). Recently, the crystal structure of murine sEH has been determined at 2.8 Å resolution (20Argiriadi M. Morisseau C. Hammock B. Christianson D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10637-10642Crossref PubMed Scopus (209) Google Scholar). The structure supports the previously proposed mechanism and suggests that Tyr465 and Tyr381are the possible acid catalysts that activate the epoxide ring (Fig.1). Additionally, analysis of the crystal structure of EH from Agrobacterium radiobacter, AD1, likewise suggested that Tyr152-Tyr215 in this prokaryotic enzyme may have the same function (21Nardini M. Ridder I.S. Rozeboom H.J. Kalk K.H. Rink R. Janssen D.B. Dijkstra B.W. J. Biol. Chem. 1999; 274: 14579-14586Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Thus, biochemical evidence is required to verify the mechanism of epoxide activation and to complement structure-based prediction. In this study, site-directed mutagenesis was utilized to explore the role of Tyr465 and Tyr381 in the catalytic mechanism of murine sEH. The sEH variants were subjected to a series of enzyme assays and kinetic studies to assess their impact on epoxide activation. Additionally, this work was extended to Tyr374of human mEH. Interpreted in view of the EH crystal structure, these results clearly support the role of active site tyrosine residues in epoxide activation by eukaryotic sEH and mEH. The following compounds were previously synthesized in our laboratory: racemic 4-nitrophenyl-2,3-epoxy-3-phenylpropyl carbonate (NEP2C, compound1) (22Dietze E.C. Kuwano E. Hammock B.D. Anal. Biochem. 1994; 216: 176-187Crossref PubMed Scopus (40) Google Scholar); [3H]trans-1,3-diphenylpropene oxide (tDPPO, compound 2); [14C]cis-9,10-epoxystearic acid (ESA, compound4) (23Borhan B. Mebrahtu T. Nazarian S. Kurth M.J. Hammock B.D. Anal. Biochem. 1995; 231: 188-200Crossref PubMed Scopus (92) Google Scholar); [3H]trans-stilbene oxide (tSO, compound 3); [3H]cis-stilbene oxide (cSO, compound 16) (24Gill S.S. Ota K. Hammock B.D. Anal. Biochem. 1983; 131: 273-282Crossref PubMed Scopus (165) Google Scholar); para-substituted chalcone oxides (compounds 6-11) (19Morisseau C. Du G. Newman J.W. Hammock B.D. Arch. Biochem. Biophys. 1998; 356: 214-228Crossref PubMed Scopus (73) Google Scholar); compounds13 and 15 (25Morisseau C. Goodrow M. Dowdy D. Zheng J. Greene J. Sanborn J. Hammock B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8849-8854Crossref PubMed Scopus (225) Google Scholar); and compound 14 (26Argiriadi M.A. Morisseau C. Goodrow M.H. Dowdy D.L. Hammock B.D. Christianson D.W. J. Biol. Chem. 2000; 275: 15265-15270Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). [3H]Juvenile hormone III (JH III; compound 5) was obtained from NEN Life Science Products. Compound 12 was purchased from Aldrich. Sequence numbers are based on the murine sEH sequence (GenBankTM/EMBL Data Bank accession number L05781) and human mEH sequence (X07936). Murine sEH cDNA or human mEH cDNA (27Grant D.F. Greene J.F. Pinot F. Borhan B. Moghaddam M.F. Hammock B.D. McCutchen B. Ohkawa H. Luo G. Guenthner T.M. Biochem. Pharmacol. 1996; 51: 503-515Crossref PubMed Scopus (22) Google Scholar) was inserted into the EcoRI or BamHI site of baculovirus expression vector, pFastBac1 (Life Technologies, Inc.). The whole plasmid was directly subjected to site-directed mutagenesis based on aPfuTurbo DNA polymerase system (Stratagene, La Jolla, CA). Oligonucleotides for murine sEH mutants were as follows: Y381F, 5′-CCAGTTTTCAATTTTCAGCTGTACTTT-3′ and 5′-AAAGTACAGCTGAAAATTGAAAACTGG-3′; Y465F, 5′-CCTCTGAACTGGTTCCGGAACACAGAA-3′ and 5′-GGAGACTTGACCAAGGCCTTGTGTCTT-3′; Y465A, 5′-CCTCTGAACTGGGGCCGGAACACAGAA-3′ and 5′-GGAGACTTGACCCCGGCCTTGTGTCTT-3′. Y381F/Y465F double mutant was constructed with the plasmid for Y465F and a set of oligonucleotides for Y381F. Oligonucleotides for human mEH mutant were as follows: Y374F, 5′-TCCCAGCGCTTCTTCAAGGAGAACCTG-3′ and 5′-CAGGTTCTCCTTGAAGAAGCGCTGGGA-3′. The mEH cDNA was obtained from Drs. C. Omiecinski and V. P. Hosagrahara (Seattle, WA). Underlined residues were modified to generate corresponding mutations. Effective mutations were verified by DNA sequencing of both strands. Enzymes were produced using BAC-TO-BAC Baculovirus Expression System (Life Technologies, Inc.). Cells from Trichoplusia ni(T. ni High5) (500 ml, 5 × 105 cells/ml) were infected with virus solution at a multiplicity of infection of 0.1. Three days postinfection, the cells were resuspended in 20 ml of 0.1 m sodium phosphate buffer (pH 7.4) (buffer A) containing 1 mm phenylmethylsulfonyl fluoride, EDTA, and dithiothreitol, and homogenized with a Polytron. The crude extract was centrifuged at 12,000 × g for 20 min. The supernatant was centrifuged again at 100,000 × g for 1 h. For murine sEH, the resulting supernatant (cytosol fraction) was stored at −80 °C, and for human mEH, the pellet (microsomal fraction) was resuspended in 3 ml of buffer A and stored at −80 °C. Protein concentrations were determined with the Pierce BCA assay (Pierce) using bovine serum albumin as a standard. SDS-polyacrylamide gel electrophoresis was performed according to Laemmli (28Laemmli U. Nature. 1970; 227: 680-685Crossref PubMed Scopus (215638) Google Scholar) using a 10% resolving gel. Assays for murine sEH, employing Western blot techniques, were performed using the corresponding polyclonal antibody as described previously (12Pinot F. Grant D.F. Beetham J.K. Parker A.G. Borhan B. Landt S. Jones A.D. Hammock B.D. J. Biol. Chem. 1995; 270: 7968-7974Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The polyclonal antibody for human mEH was a gift of Drs. Franz Oesch and Michael Arand (Mainz, Germany). Estimation of protein bands was carried out with the Scion Image software package (Scion, Frederick, MD). The sEH enzyme activity of the sEH wild type and its mutants was determined using five different substrates (compounds 1-5). These assays were performed as described previously: NEP2C (1) (19Morisseau C. Du G. Newman J.W. Hammock B.D. Arch. Biochem. Biophys. 1998; 356: 214-228Crossref PubMed Scopus (73) Google Scholar), tDPPO (2) (23Borhan B. Mebrahtu T. Nazarian S. Kurth M.J. Hammock B.D. Anal. Biochem. 1995; 231: 188-200Crossref PubMed Scopus (92) Google Scholar), tSO (3) (24Gill S.S. Ota K. Hammock B.D. Anal. Biochem. 1983; 131: 273-282Crossref PubMed Scopus (165) Google Scholar), [14C]ESA (4) (23Borhan B. Mebrahtu T. Nazarian S. Kurth M.J. Hammock B.D. Anal. Biochem. 1995; 231: 188-200Crossref PubMed Scopus (92) Google Scholar), and JH III (5) (23Borhan B. Mebrahtu T. Nazarian S. Kurth M.J. Hammock B.D. Anal. Biochem. 1995; 231: 188-200Crossref PubMed Scopus (92) Google Scholar, 29Mumby S.M. Hammock B.D. Anal. Biochem. 1979; 92: 16-21Crossref PubMed Scopus (44) Google Scholar). The assays were run at 30 °C for 1–30 min depending on the substrate and the mutant used. To compare specific activities between wild-type and the mutant enzymes, identical concentrations of enzymes were used as judged by Western blot ([E] = 80 nm for 1, 3, 4, 5 and 3 nm for 2). For the human microsomal EH, cSO (compound 16) was used as substrate. Assay was performed as described (24Gill S.S. Ota K. Hammock B.D. Anal. Biochem. 1983; 131: 273-282Crossref PubMed Scopus (165) Google Scholar) using 1 μg of the microsomal fraction. The wild-type enzyme was incubated at 30 °C for 1 min, while the Y374F mutant was incubated for 30 min. Cytosol fractions of control cells have no detectable epoxide hydrolase activities above background (30Beetham J.K. Tian T. Hammock B.D. Arch. Biochem. Biophys. 1993; 305: 197-201Crossref PubMed Scopus (147) Google Scholar). The kinetic constants were determined using the two-step inhibition model previously described for EH inhibition, where chalcone oxides and NEP2C were used as substrate (19Morisseau C. Du G. Newman J.W. Hammock B.D. Arch. Biochem. Biophys. 1998; 356: 214-228Crossref PubMed Scopus (73) Google Scholar). Inhibitor concentrations between 0 and 2.5 μm were used for the wild-type enzyme, whereas concentrations between 0 and 50 μm were used for the two mutant enzymes. To achieve homogeneity in the results and to be able to compare them between the wild-type and the two mutants, Y381F and Y465F, identical concentrations of enzymes (80 nm) were used as determined by Western blot. Measurements were made in the presence of excess of substrate ([S] = 40 μm). Activity measurements at 0–30 s post inhibitor introduction allow determination of the initial rate of enzyme inhibitor complex formation (ρ) as described previously (19Morisseau C. Du G. Newman J.W. Hammock B.D. Arch. Biochem. Biophys. 1998; 356: 214-228Crossref PubMed Scopus (73) Google Scholar). Under these conditions, less than 10% of the inhibitor was bound to the enzyme. The enzyme-inhibitor dissociation constant (Kd) and the rate of enzyme-inhibitor covalent complex formation (k 2) were calculated from plots of ρ versus [I] (19Morisseau C. Du G. Newman J.W. Hammock B.D. Arch. Biochem. Biophys. 1998; 356: 214-228Crossref PubMed Scopus (73) Google Scholar). The rates of dealkylation and product release (k 3) were calculated from enzyme activities in 1:1 enzyme:inhibitor mixtures, at times varying between 1 and 20 min post inhibitor introduction as described previously (19Morisseau C. Du G. Newman J.W. Hammock B.D. Arch. Biochem. Biophys. 1998; 356: 214-228Crossref PubMed Scopus (73) Google Scholar). The decomposition rate of the inhibitors in the absence of the enzyme was found to be negligible (<1%) over the period studied. The accuracy of the applied kinetic model was assessed by comparing theoretical and measured results for ρ, producing correlation factors greater than 0.95. Based on experimental inhibitor and substrate concentrations, a maximal error in observedKd of ∼5% can be attributed to incomplete dissociation of EI. Moreover, the k 2and k 3 values obtained indicated a 7% maximal error in k 2 because of the ECdegradation. IC50 values were determined using tDPPO (compound 2) as a substrate. Enzymes (3 nm) were incubated with inhibitors for 5 min at 30 °C prior to substrate introduction ([S] = 50μ m). Assays were run for 1 min for wild-type and were extended to 15 min for Y381F and Y465F. IC50 was calculated by linear regression of five datum points with a minimum of two points of either side of IC50. The results were generated from at least three separate runs each in triplicate to obtain the standard deviation. Images used in this study were rendered and displayed in the Swiss-Pdb Viewer (31Guex N. Experientia (Basel). 1996; 52 (abstr.): 26Google Scholar, 32Guex N. Peitsch M.C. Protein Data Bank Quarterly Newsletter. 1996; 77: 7Google Scholar, 33Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9793) Google Scholar) using coordinates supplied by Argiriadi et al. (26Argiriadi M.A. Morisseau C. Goodrow M.H. Dowdy D.L. Hammock B.D. Christianson D.W. J. Biol. Chem. 2000; 275: 15265-15270Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Additional graphic enhancements were performed in the Photoshop graphic programming environment. To explore the role of each of the tyrosines of murine sEH, we prepared four different mutant constructs, Y381F, Y465F, Y465A, and Y381F/Y465F. The enzymes were subsequently produced using baculovirus expression system as described under “Experimental Procedures.” In the purification of murine sEH, the ligand (Fig.2 A) interacts with the putative hydrophobic pocket near the active site (12Pinot F. Grant D.F. Beetham J.K. Parker A.G. Borhan B. Landt S. Jones A.D. Hammock B.D. J. Biol. Chem. 1995; 270: 7968-7974Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 34Wixtrom R.N. Silva M.H. Hammock B.D. Anal. Biochem. 1988; 169: 71-80Crossref PubMed Scopus (86) Google Scholar). Wild-type and mutant protein were detected at similar expression level in the cytosol fraction and not detected in the flow through fraction (Fig.2 C) indicating that the mutant enzyme probably maintained its structural integrity. The mutant enzymes were eluted in low yield with 1 mm 4-fluorochalcone oxide (FCO), a selective inhibitor for sEH, indicating probable lower affinity for the inhibitor (Fig. 2, D–E). Total protein eluted by FCO followed by SDS (Fig. 2 E) was similar for both wild and mutant enzymes, indicating same overall binding for each. The specific activities of the wild-type and mutant enzymes in the hydrolysis of five substrates are summarized in TableI. These substrates are classified, and ordered from the more reactive (NEP2C (compound 1)) to the less reactive (JH III (compound 5)). The specific activities of wild-type enzyme were similar to previously published results (19Morisseau C. Du G. Newman J.W. Hammock B.D. Arch. Biochem. Biophys. 1998; 356: 214-228Crossref PubMed Scopus (73) Google Scholar,23Borhan B. Mebrahtu T. Nazarian S. Kurth M.J. Hammock B.D. Anal. Biochem. 1995; 231: 188-200Crossref PubMed Scopus (92) Google Scholar). NEP2C and JH III are known to be hydrolyzed by esterases; however, the esterase-dependent hydrolysis was not detectable in these experiments (data not shown). In mutant enzymes, the activities were lower than those of the wild-type. Substrates containing less reactive epoxides resulted in larger changes in activity between the wild-type and the mutant enzymes, suggesting that hydrolysis of less reactive epoxides is dependent on activation by tyrosine(s). The Y381F/Y465F mutant demonstrated no detectable catalytic activity with any of the substrates used. These results suggest that both Tyr381 and Tyr465 are required for full activity of the murine sEH. This is consistent with the results of analogous experiments with bacterial EH showing that active site tyrosines Tyr152 and Tyr215 are important for catalysis (35Rink R. Spelberg J.H.L. Pieters R.J. Kingma J. Nardini M. Kellogg R.M. Dijkstra B.W. Janssen D.B. J. Am. Chem. Soc. 1999; 121: 7417-7418Crossref Scopus (65) Google Scholar).Table ISpecific activities of wild-type and mutant murine sEHAll assays were performed on the cytosol fraction from baculovirus-infected T. ni cells. The reported results are the mean ± S.D. (n = 3). Levels of sEH protein were apparently identical in all assays based on Western blot.1-a n.d., nondetectable. Open table in a new tab All assays were performed on the cytosol fraction from baculovirus-infected T. ni cells. The reported results are the mean ± S.D. (n = 3). Levels of sEH protein were apparently identical in all assays based on Western blot. 1-a n.d., nondetectable. To further investigate the role of the two residues Tyr381 and Tyr465 in the catalytic mechanism, we determined the kinetic constants of their inhibition by chalcone oxides. Chalcone oxides are in fact poor EH substrates that inhibit the enzyme by forming a stable covalent intermediate (19Morisseau C. Du G. Newman J.W. Hammock B.D. Arch. Biochem. Biophys. 1998; 356: 214-228Crossref PubMed Scopus (73) Google Scholar). Their action is described by the following equation.E+I⇄KdEI→k2EC→k3E+PEquation 1 The nonlinear regression of the initial rate of ECformation (ρ) versus inhibitor concentration ([I]) permits the calculation of Kd and k 2 (19Morisseau C. Du G. Newman J.W. Hammock B.D. Arch. Biochem. Biophys. 1998; 356: 214-228Crossref PubMed Scopus (73) Google Scholar). The slope of the EC complex decomposition, ln(A0 − A) versus time, permits the calculation of k 3 (19Morisseau C. Du G. Newman J.W. Hammock B.D. Arch. Biochem. Biophys. 1998; 356: 214-228Crossref PubMed Scopus (73) Google Scholar). Derived kinetic constants are listed in Table II.Table IIKinetic constants for inhibition of wild-type and mutant murine sEH by chalcone oxides2-a Value ± 95% confidence interval.2-b Mean ± S.D. (n = 3). Open table in a new tab 2-a Value ± 95% confidence interval. 2-b Mean ± S.D. (n = 3). Both mutations of Tyr465 and of Tyr381 by phenylalanine resulted in increases in the Kd values up to 50-fold, with the exception of compound 7 and the Tyr381 mutant (Table II). These results demonstrate that both tyrosines are implicated in the binding of the substrate. The change in Kd is well correlated (r2 = 0.91) with the ς values of the para-substitution of chalcone oxides for Y465F, whereas no relationships were found for Y381F. The observed increased Kd in mutant enzymes reflect the smaller interaction between the enzyme and the inhibitor. The linear relation observed between Kd and ς for Y465F highlights the possible bond between Tyr465 and epoxide moiety, as stated (26Argiriadi M.A. Morisseau C. Goodrow M.H. Dowdy D.L. Hammock B.D. Christianson D.W. J. Biol. Chem. 2000; 275: 15265-15270Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The rate of alkylation (k 2) is also greatly altered in both mutations (Table II). The values of k 2 for Y465F decreased increasingly from 2- to 30-fold for compounds 11 to 6, respectively. For Y381F such trends in the decreased k 2 were observed only from compound 9 to 6, whereas compounds 11 to 9 showed a 7-fold decrease in their k 2 values. The Hammett plot (Fig.3) showing the logarithm of the relative rates (4−R k 2/4− H k 2) versus the constant ς+ for each 4-position substituent illustrates this influence. These results were correlated with ς+ for the three enzymes, indicating that development of a relative positive charge at the reactive center is important for the alkylation step. For the wild-type enzyme, a linear relationship (r2 = 0.93) with a slope of −0.56 was obtained, indicating a push-pull mechanism, in which the epoxide oxygen is activated by protonation facilitating a nucleophilic attack on the carbon of the epoxide ring by the Asp333 carboxylate anion (19Morisseau C. Du G. Newman J.W. Hammock B.D. Arch. Biochem. Biophys. 1998; 356: 214-228Crossref PubMed Scopus (73) Google Scholar). A linear relationship (r2 = 0.91) was obtained for Y465F, indicating a consistent mechanism operating throughout the chalcone oxide series. However, the sign of the slope (+ 0.27) is inverted from the slope of the of the wild-type enzyme, indicating a different mechanism. This value is very close to the value (+0.32) found for a general basic mechanism of opening of similar epoxides (36Blumenstein J. Ukachukwu V. Mohan R. Whaleen D. J. Org. Chem. 1993; 58: 924-932Crossref Scopus (51) Google Scholar). This result strongly suggests that a simple nucleophilic mechanism is implied in the action of Y465F and that Tyr465is directly related to the polarization of the epoxide moiety. A bell shaped relationship was obtained for Y381F, indicating a change of mechanism operating throughout the chalcone oxide series. A slope (+0.26) similar to one of Y465F is obtained for the electron donatingpara-substitutions (6 to 9), whereas a slope (−0.51) similar to the one for wild type is obtained for electron withdrawing para-substitutions (9 to11). Therefore, Tyr381 participates in the polarization of the epoxide moiety. However, its role is less clear than that of Tyr465. The rate of dealkylation k 3 was less influence by either of the tyrosine mutations; the decreasedk 3 between 1- and 3-fold are observed for Y465F and Y381F compared with the wild type (Table II). Moreover, no relation was found between the intensity of the change in k 3 and the nature of the para-substitution for Y465F and Y381F. These results indicate that neither tyrosine directly influences the hydrolysis of the enzyme-inhibitor covalent intermediate. This agrees with prediction from the crystal structure (20Argiriadi M. Morisseau C. Hammock B. Christianson D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10637-10642Crossref PubMed Scopus (209) Google Scholar). Additionally, changes in specific activities for compounds 1-5 probably came from the changes in Kd and k 2, because k 3 was unchanged for compounds6-11. The crystal structure of sEH-N-cyclohexyl-N′-decyl urea (compound 14) complex shows that Tyr465 and Tyr381 provide hydrogen bond interaction with the carbonyl group of the urea (26Argiriadi M.A. Morisseau C. Goodrow M.H. Dowdy D.L. Hammock B.D. Christianson D.W. J. Biol. Chem. 2000; 275: 15265-15270Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). To evaluate this apparent binding trend with the tyrosines, IC50 values were determined for wild type and for both tyrosine mutants (TableIII). Substitution of Tyr381by phenylalanine resulted in an enzyme with 8–88-fold higher IC50 values for compounds tested. Replacement of Tyr465 by phenylalanine increased IC50 by 2–13-fold. These results suggest that both Tyr381 and Tyr465 interact with these inhibitors. Particularly Tyr381