Leukotriene A4 Hydrolase

Leukotriene (LT) A4 hydrolase is a bifunctional zinc metalloenzyme, which converts LTA4 into the neutrophil chemoattractant LTB4 and also exhibits an anion-dependent aminopeptidase activity. In the x-ray crystal structure of LTA4 hydrolase, Arg563 and Lys565 are found at the entrance of the active center. Here we report that replacement of Arg563, but not Lys565, leads to complete abrogation of the epoxide hydrolase activity. However, mutations of Arg563 do not seem to affect substrate binding strength, because values of Ki for LTA4 are almost identical for wild type and (R563K)LTA4 hydrolase. These results are supported by the 2.3-Å crystal structure of (R563A)LTA4 hydrolase, which does not reveal structural changes that can explain the complete loss of enzyme function. For the aminopeptidase reaction, mutations of Arg563 reduce the catalytic activity (Vmax = 0.3–20%), whereas mutations of Lys565 have limited effect on catalysis (Vmax = 58–108%). However, in (K565A)- and (K565M)LTA4 hydrolase, i.e. mutants lacking a positive charge, values of the Michaelis constant for alanine-p-nitroanilide increase significantly (Km = 480–640%). Together, our data indicate that Arg563 plays an unexpected, critical role in the epoxide hydrolase reaction, presumably in the positioning of the carboxylate tail to ensure perfect substrate alignment along the catalytic elements of the active site. In the aminopeptidase reaction, Arg563 and Lys565 seem to cooperate to provide sufficient binding strength and productive alignment of the substrate. In conclusion, Arg563 and Lys565 possess distinct roles as carboxylate recognition sites for two chemically different substrates, each of which is turned over in separate enzymatic reactions catalyzed by LTA4 hydrolase. Leukotriene (LT) A4 hydrolase is a bifunctional zinc metalloenzyme, which converts LTA4 into the neutrophil chemoattractant LTB4 and also exhibits an anion-dependent aminopeptidase activity. In the x-ray crystal structure of LTA4 hydrolase, Arg563 and Lys565 are found at the entrance of the active center. Here we report that replacement of Arg563, but not Lys565, leads to complete abrogation of the epoxide hydrolase activity. However, mutations of Arg563 do not seem to affect substrate binding strength, because values of Ki for LTA4 are almost identical for wild type and (R563K)LTA4 hydrolase. These results are supported by the 2.3-Å crystal structure of (R563A)LTA4 hydrolase, which does not reveal structural changes that can explain the complete loss of enzyme function. For the aminopeptidase reaction, mutations of Arg563 reduce the catalytic activity (Vmax = 0.3–20%), whereas mutations of Lys565 have limited effect on catalysis (Vmax = 58–108%). However, in (K565A)- and (K565M)LTA4 hydrolase, i.e. mutants lacking a positive charge, values of the Michaelis constant for alanine-p-nitroanilide increase significantly (Km = 480–640%). Together, our data indicate that Arg563 plays an unexpected, critical role in the epoxide hydrolase reaction, presumably in the positioning of the carboxylate tail to ensure perfect substrate alignment along the catalytic elements of the active site. In the aminopeptidase reaction, Arg563 and Lys565 seem to cooperate to provide sufficient binding strength and productive alignment of the substrate. In conclusion, Arg563 and Lys565 possess distinct roles as carboxylate recognition sites for two chemically different substrates, each of which is turned over in separate enzymatic reactions catalyzed by LTA4 hydrolase. The leukotrienes (LTs) 1The abbreviations used are: LT, leukotriene; LTA4H, leukotriene A4 hydrolase; pNA, p-nitroanilide.1The abbreviations used are: LT, leukotriene; LTA4H, leukotriene A4 hydrolase; pNA, p-nitroanilide. are a class of structurally related lipid mediators involved in the development and maintenance of inflammatory and allergic reactions (1Samuelsson B. Dahlén S.-E. Lindgren J.-Å. Rouzer C.A. Serhan C.N. Science. 1987; 237: 1171-1176Crossref PubMed Scopus (1960) Google Scholar, 2Funk C.D. Science. 2001; 294: 1871-1875Crossref PubMed Scopus (2966) Google Scholar). In the biosynthesis of LTs, 5-lipoxygenase converts arachidonic acid into the unstable epoxide LTA4. This intermediate may in turn be conjugated with glutathione to form the spasmogenic LTC4, or hydrolyzed into the proinflammatory lipid mediator LTB4,ina reaction catalyzed by LTA4 hydrolase (LTA4H). Leukotriene B4 is a classical chemoattractant of human neutrophils and triggers adherence and aggregation of leukocytes to vascular endothelium at only nanomolar concentrations (3Samuelsson B. Science. 1983; 220: 568-575Crossref PubMed Scopus (2307) Google Scholar). In addition, LTB4 modulates immune responses (4Payan D.G. Missirian-Bastian A. Goetzl E.J. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 3501-3505Crossref PubMed Scopus (110) Google Scholar, 5Yamaoka K.A. Claesson H.-E. Rosén A. J. Immunol. 1989; 143: 1996-2000PubMed Google Scholar) and recent studies show that this lipid mediator is involved in early effector CD4+ and CD8+ T cell recruitment to sites of inflammation (6Tager A.M. Bromley S.K. Medoff B.D. Islam S.A. Bercury S.D. Friedrich E.B. Carafone A.D. Gerszten R.E. Luster A.D. Nat. Immunol. 2003; 4: 982-990Crossref PubMed Scopus (335) Google Scholar, 7Ott V.L. Cambier J.C. Kappler J. Marrack P. Swanson B.J. Nat. Immunol. 2003; 4: 974-981Crossref PubMed Scopus (233) Google Scholar, 8Goodarzi K. Goodarzi M. Tager A.M. Luster A.D. von Andrian U.H. Nat. Immunol. 2003; 4: 965-973Crossref PubMed Scopus (287) Google Scholar). Moreover, LTB4 participates in the host-defense against infections (9Bailie M.B. Standiford T.J. Laichalk L.L. Coffey M.J. Strieter R. Peters-Golden M. J. Immunol. 1996; 157: 5221-5224PubMed Google Scholar, 10Mancuso P. Nana-Sinkam P. Peters-Golden M. Infect. Immun. 2001; 69: 2011-2016Crossref PubMed Scopus (110) Google Scholar) and is a key mediator of platelet activating factor-induced lethal shock (11Chen X.S. Sheller J.R. Johnson E.N. Funk C.D. Nature. 1994; 372: 179-182Crossref PubMed Scopus (351) Google Scholar, 12Byrum R.S. Goulet J.L. Griffiths R.J. Koller B.H. J. Exp. Med. 1997; 185: 1065-1075Crossref PubMed Scopus (116) Google Scholar, 13Byrum R.S. Goulet J.L. Snouwaert J.N. Griffiths R.J. Koller B.H. J. Immunol. 1999; 163: 6810-6819PubMed Google Scholar). These effects are signaled via specific, G protein-coupled receptors for LTB4 (BLT1 and BLT2) (14Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (845) Google Scholar, 15Yokomizo T. Kato K. Terawaki K. Izumi T. Shimizu T. J. Exp. Med. 2000; 192: 421-431Crossref PubMed Scopus (466) Google Scholar). The relative contributions of these two receptors to signaling and bioactivity are presently not clear. LTA4H (EC 3.3.2.6) is a bifunctional zinc metalloenzyme, exhibiting an anion-dependant aminopeptidase activity in addition to its epoxide hydrolase activity, i.e. the hydrolysis of the unstable allelic epoxide LTA4 into LTB4, for a review, see Ref. 16Haeggström J.Z. Am. J. Resp. Crit. Care Med. 2000; 161: 25-31Crossref PubMed Scopus (67) Google Scholar. Unlike the aminopeptidase activity, the epoxide hydrolase activity is restrained by suicide inactivation that involves binding of LTA4 to Tyr378, which is located within a 21-residue active-site peptide denoted K21 (17Mueller M.J. Wetterholm A. Blomster M. Jörnvall H. Samuelsson B. Haeggström J.Z. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8383-8387Crossref PubMed Scopus (49) Google Scholar, 18Mueller M.J. Blomster M. Oppermann U.C. Jörnvall H. Samuelsson B. Haeggström J.Z. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5931-5935Crossref PubMed Scopus (48) Google Scholar). The epoxide hydrolase reaction of LTA4H is also unique in the sense that the stereoselective introduction of water to the carbon backbone of LTA4 occurs several methylene units away from the epoxide moiety, presumably proceeding via a carbocation intermediate (19Blomster Andberg M. Hamberg M. Haeggström J.Z. J. Biol. Chem. 1997; 272: 23057-23063Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Furthermore, Asp375 was shown to assist in the introduction of the 12R hydroxyl group of LTB4 (20Rudberg P.C. Tholander F. Thunnissen M.M. Samuelsson B. Haeggström J.Z. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4215-4220Crossref PubMed Scopus (36) Google Scholar), a key component for the biologic activity of LTB4 (21Ford-Hutchinson A.W. Bray M.A. Cunningham F.M. Davidson E.M. Smith M.J. Prostaglandins. 1981; 21: 143-152Crossref PubMed Scopus (59) Google Scholar, 22Leblanc Y. Fitzsimmons B.J. Charleson S. Alexander P. Evans J.F. Rokach J. Prostaglandins. 1987; 33: 617-625Crossref PubMed Scopus (25) Google Scholar, 23Yokomizo T. Kato K. Hagiya H. Izumi T. Shimizu T. J. Biol. Chem. 2001; 276: 12454-12459Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Certain arginyl di- and tripeptides as well as p-nitroanilide derivatives of Ala and Arg are very efficient substrates for the aminopeptidase activity (24Örning L. Gierse J.K. Fitzpatrick F.A. J. Biol. Chem. 1994; 269: 11269-11273Abstract Full Text PDF PubMed Google Scholar). Although it has never been proven, it is generally believed that the peptidase activity is involved in the processing of bioactive peptides related to inflammation and host defense. Several lines of evidence indicate that the aminopeptidase activity follows a zinc-assisted general base mechanism with Glu296 and Tyr383 acting as the general base and proton donor, respectively. Moreover, in recent studies, Glu271 was identified as the recognition site for the N-terminal amino group of the peptidase substrate (25Rudberg P.C. Tholander F. Thunnissen M.M. Haeggström J.Z. J. Biol. Chem. 2002; 277: 1398-1404Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Recently, we solved the crystal structure of LTA4H in complex with the aminopeptidase inhibitor bestatin at 1.95-Å resolution, which has given important clues to the molecular mechanisms of the two enzyme reactions (26Thunnissen M.M. Nordlund P. Haeggström J.Z. Nat. Struct. Biol. 2001; 8: 131-135Crossref PubMed Scopus (254) Google Scholar). Earlier work with chemical modification already indicated the presence of essential Arg residues near or at the active center of LTA4H (27Mueller M.J. Samuelsson B. Haeggström J.Z. Biochemistry. 1995; 34: 3536-3543Crossref PubMed Scopus (18) Google Scholar). From the x-ray crystal structure of LTA4H, a positively charged site was identified in a cavity formed between the N-terminal, the central catalytic, and the C-terminal domain (Fig. 1). This site, composed of Arg563 and Lys565, is located in a wide portion of the cavity, near its entrance, and in the vicinity of the carboxyl group of bestatin. These two residues are positioned in the first turn of an α-helix pointing toward the active site. Whereas Arg563 is buried, Lys565 is fully exposed to solvent. Also, when LTA4 was modeled into the active site, electrostatic interactions seemed possible between the C1 carboxylate of LTA4 and the positively charged side groups of Arg563 and/or Lys565 (Fig. 1). Furthermore, in the crystal structure of LTA4H in complex with a specific hydroxamic acid inhibitor that is a structural mimic of LTA4, a direct interaction between the carboxylic moiety of this inhibitor and Arg563 is seen (28Thunnissen M.M. Andersson B. Samuelsson B. Wong C.H. Haeggström J.Z. FASEB J. 2002; 16: 1648-1650Crossref PubMed Scopus (65) Google Scholar). In this report we used site-directed mutagenesis combined with x-ray crystallography and inhibition studies to analyze the functional role of Arg563 and Lys565 as putative carboxylate recognition sites in LTA4H. We show that Arg563 is required for the epoxide hydrolase activity, presumably in an indirect manner to achieve correct alignment of LTA4 along the catalytic elements of the active site. As a result, Arg563 is a key residue during LTB4 formation. For the aminopeptidase reaction, we propose that Lys565 assists Arg563 for optimal substrate binding. Thus, we have identified a common carboxylate recognition site for lipid and peptide substrates in the active center of LTA4H. Materials—Oligonucleotides were synthesized by Cybergene, Stockholm, Sweden. LTA4 methyl ester was synthesized as described (29Ollmann I.R. Hogg J.H. Munoz B. Haeggström J.Z. Samuelsson B. Wong C.-H. Bioorg. Med. Chem. 1995; 3: 969-995Crossref PubMed Scopus (25) Google Scholar) or purchased from BIOMOL Research Laboratories Plymouth Meeting, PA. LTA4 methyl ester was saponified in tetrahydrofuran with 1 m LiOH (6% v/v) for 48 h at 4 °C. Alanine-p-nitroanilide, isopropyl-β-d-thiogalactopyranoside, phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, and streptomycin sulfate were purchased from Sigma. Nickel-nitrilotriacetic acid resin was from Qiagen. Mutagenesis of Human LTA4H cDNA—Site-directed mutagenesis was carried out by PCR on the recombinant plasmid pT3MB5 for expression of (His)6-tagged LTA4H in Escherichia coli. This plasmid is a variant of pT3MB4 (18Mueller M.J. Blomster M. Oppermann U.C. Jörnvall H. Samuelsson B. Haeggström J.Z. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5931-5935Crossref PubMed Scopus (48) Google Scholar) in which an NsiI restriction site has been eliminated by a single nucleotide exchange. Briefly, site-directed mutagenesis involves two consecutive steps of PCR comprising four different primers, according to the megaprimer method (30Sarkar G. Sommer S.S. BioTechniques. 1990; 8: 404-407PubMed Google Scholar). Polymerase chain reactions were carried out in a total volume of 50 μl, using 1× Pfu polymerase buffer, 125–150 ng each of primers A and B, 1 unit of Pfu polymerase, 10 nmol each of dNTPs, and 100 ng of template. In the second reaction, 15–20 μl of the first PCR mixture was used to supply the reaction with the megaprimer, which was used together with primer C (125–150 ng). The amplification program included an initial round of denaturation at 94 °C (60 s), annealing at 55–66 °C (60 s), and elongation at 72 °C (90 s), followed by 30 cycles of denaturation (45 s), annealing (30 s), and elongation (60 s) on a PE GeneAmp PCR System 2400. For generation of (E560Q)-, (R563K)-, (R563A)-, (R563M)-, (K565R)-, (K565A)-, (K565M)-, and (R563K/K565R)LTA4H, BfrI and NsiI sites were used. DNA fragments were cleaved with BfrI/NsiI and purified by agarose gel electrophoresis (1.5%) followed by extraction (QIAEX II Gel Extraction Kit). Mutated fragments were ligated into pT3MB5 (T4 DNA Ligase Protocol), opened with the same restriction enzymes. Competent E. coli cells (JM101) were transformed with mutated recombinant plasmid and grown in LB medium containing ampicillin (100 μg/ml). Stock cultures were kept at -70 °C in a 1:1 mixture of culture medium and 40% (v/v) glycerol, 0.75% (w/v) NaCl, respectively. Recombinant plasmids were purified using Wizard Minipreps Plus, and the entire mutated inserts were all sequenced using Dynamic ET Terminator Cycle Sequencing Kit (Amersham Biosciences) to confirm that no nucleotide alterations had occurred in addition to the desired mutation. Protein Expression and Purification—Mutated enzymes were expressed as N-terminal (His)6-tag fusion proteins in E. coli (JM101) cells grown at 37 °C in M9 medium (50 mm Na2HPO4, 22 mm KH2PO4, 20 mm NH4Cl, 8.5 mm NaCl), pH 7.4, containing 0.4% glucose (w/v), 0.2% (w/v) casamino acids, 2 mm MgSO4, and 0.1 mm CaCl2. At A620 ≈ 0.2, isopropyl-β-d-thiogalactopyranoside was added to a final concentration of 500 μm. Cells were harvested at A620 ≈ 1.8, pelleted at 1000 × g, and resuspended in 30 ml of homogenization buffer (50 mm Tris-HCl, pH 8.0, containing soybean trypsin inhibitor) supplemented with phenylmethylsulfonyl fluoride (1 mm). Nucleic acids were removed by streptomycin sulfate precipitation. After centrifugation (10,000 × g for 15 min), the supernatant was filtered (0.22 μm) and applied to a nickelnitrilotriacetic acid resin. The column was washed with 1 bed volume of 50 mm Tris-HCl, pH 8.0, 50 mm sodium phosphate buffer, pH 6.8, containing 0.5 m NaCl, and 50 mm Tris-HCl, pH 8.0, with each solution supplemented with 10 mm imidazole. The His-tagged protein was eluted with 1.4 bed volumes of 50 mm Tris-HCl, pH 8.0, containing 100 mm imidazole. The purity of the final preparation was assessed by SDS-PAGE on a Pharmacia Phast system, using 10–15% gradient gels, subsequently stained with Coomassie Brilliant Blue. Protein concentrations were determined by the Bradford method using a MCC/340 96-well multiscan spectrophotometer and bovine serum albumin as standard (31Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar). Aminopeptidase Activity Assays—The aminopeptidase activity, expressed as Vmax, was determined in a spectrophotometric assay at 405 nm, using a MCC/340 multiscan spectrophotometer, essentially as described (32Wetterholm A. Haeggström J.Z. Samuelsson B. Yuan W. Munoz B. Wong C.-H. J. Pharmacol. Exp. Ther. 1995; 275: 31-37PubMed Google Scholar). Briefly, the enzyme (1–20 μg) was incubated at room temperature in 96-well microtiter plates with alanine-p-nitroanilide as substrate in 50 mm Tris-HCl, pH 8.0, containing 100 mm KCl. The absorbance at 405 nm was measured at 10-min intervals. Different concentrations of substrate (0.125, 0.25, 0.5, 1, 2, 4, and 8 mm) were used for kinetic determinations. Spontaneous hydrolysis of the substrate was corrected by subtracting the absorbance of blank incubations without enzyme. The Michaelis constant (Km) and Vmax were determined by plotting the calculated velocity as a function of substrate concentration, whereby a nonlinear regression analysis was performed. Epoxide Hydrolase Activity Assays and Reverse-phase High Performance Liquid Chromatography—The epoxide hydrolase activity, expressed as Vmax, was determined from incubations of enzyme (1–20 μg) in 100 μl of 10 mm Tris-HCl, pH 8.0, with LTA4 (2.5–125 μm) for 30 s on ice. Reactions were quenched with 200 μl of MeOH, followed by the addition of 0.4 nmol of prostaglandin B1 or prostaglandin B2 as internal standards. Samples were acidified with 5 μl of acetic acid (10%), and metabolites were extracted on solid phase Chromabond C18 columns. Metabolites of LTA4 were separated by isocratic reverse-phase high performance liquid chromatography on a Waters Nova-Pak C18 column eluted with a mixture of methanol/acetonitrile/water/acetic acid (30:30: 40:0.01, v/v) at a flow rate of 1.2 ml/min. The UV detector was set at 270 nm and metabolites were quantified using the program Chromatography Station for Windows version 1.7. Calculations were based on peak area measurements and the known extinction coefficients for the internal standards prostaglandin B1 and prostaglandin B2 (30,000 m-1 × cm-1) as well as LTB4 (50,000 m-1 × cm-1). The Michaelis constant (Km) and Vmax were determined by plotting the calculated velocity as a function of substrate concentration, whereby a non-linear regression analysis was performed. Inhibition Assays—Prior to performing aminopeptidase activity assays, wild type-, (R563K)- and (R563M)LTA4H were preincubated with a hydroxamic acid inhibitor, 0.01–0.3 μm,orLTA4,1–10 μm. Aminopeptidase assays were then performed as described. Crystallization—Plate-like crystals of (R563A)LTA4H were obtained by liquid-liquid diffusion in capillaries, as previously described (26Thunnissen M.M. Nordlund P. Haeggström J.Z. Nat. Struct. Biol. 2001; 8: 131-135Crossref PubMed Scopus (254) Google Scholar). Briefly, 5 μl of precipitation solution (28% (v/v) PEG8000, 0.1 mm sodium acetate, 0.1 mm imidazole buffer, pH 6.8, and 5 mm YbCl3) was injected into the bottom of a melting point capillary and an equal volume of (R563A)LTA4H (5 mg/ml) in 10 mm Tris-HCl, pH 8, containing 1 mm bestatin, was layered on top. Data Collection and Structure Determination—For data collection, crystals were soaked in 15% (w/v) PEG8000, 50 mm sodium acetate, 50 mm imidazole buffer, pH 6.8, 2.5 mm YbCl3, and 25% (v/v) glycerol. Data were collected at beamline I711 of Max-Lab, Lund, Sweden. A complete set of data was collected from a single crystal. Statistics on data collection and quality are given in Table I. The crystals belonged to space group P212121 with cell dimensions a = 78, b = 86.85, c = 98.95, α = β = γ = 90° at 100 K. Processing, scaling, and merging of data were carried out using the program Mosflm (33Leslie A.G.W. Joint CCP4 and ESF-EACMB Newsletter on Protein Crystallography. 1992; Google Scholar) and programs from CCP4 (34Collaborative Computing Project umber 4Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19668) Google Scholar). The (E271Q)LTA4H mutant structure (25Rudberg P.C. Tholander F. Thunnissen M.M. Haeggström J.Z. J. Biol. Chem. 2002; 277: 1398-1404Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) was used as the starting point for refinement. All refinement was done using the CNS package (35Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D. 1998; 54: 905-921Crossref PubMed Scopus (16919) Google Scholar). Manual model building as well as interpretation of electron density maps were performed using the program XtalView (36McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2016) Google Scholar). During refinement, 614 water molecules, one Zn2+ ion, one imidazole molecule, one acetate molecule, and one Yb3+ ion were identified. The final R-factor was 18.8% and the Rfree factor was 23.9%. 3.3% of total reflections were set aside to calculate the Rfree. Most of the model of (R563A)LTA4H is in good density except for the (His)6 tag and the first four N-terminal residues. In the model, 99.4% of the residues are confined to the most favorable or additionally allowed regions and 0.6% in the generously allowed regions of the Ramachandran plot. Root mean square deviations for bond lengths and angles were 0.0079 Å and 1.44°.Table IData collection and refinement statistics Data for the highest resolution shell (2.3–2.42 Å) are shown within parantheses. Coordinates of [R563A]LTA4H have been deposited in the Protein Data Bank with accession code 1SQM.Data collection statistics Diffraction limit (Å)2.3 Wavelength (Å)0.968 Completeness (%)99.7 (99.7) Mean I/σ (I)5.3 (3) Multiplicity of observation4.7 (4.7) RmergeaRmerge = ΣhΣiIi (h) – I(h) / ΣhΣiIi (h), where Ii (h) is the ith measurement of reflection h and I(h) is the weighted mean of all measurements of h (%)9.2 (23.1)Refinement statistics R-factorbR-factor = ΣhFobs (h) – Fcalc (h) / ΣhFobs (h), where Fobs and Fcalc are the observed and calculated factor amplitudes, respectively (%)18.8 RfreecRfree is the R-factor calculated for the test set of reflections (3.3%) that are omitted during the refinement process (%)23.9 Root mean square deviations in bond distance (Å)0.0079 Root mean square deviations in bond angle (°)1.44a Rmerge = ΣhΣiIi (h) – I(h) / ΣhΣiIi (h), where Ii (h) is the ith measurement of reflection h and I(h) is the weighted mean of all measurements of hb R-factor = ΣhFobs (h) – Fcalc (h) / ΣhFobs (h), where Fobs and Fcalc are the observed and calculated factor amplitudes, respectivelyc Rfree is the R-factor calculated for the test set of reflections (3.3%) that are omitted during the refinement process Open table in a new tab Mutagenetic Replacements, Expression, and Purification of Recombinant Proteins—To detail the function of the Arg/Lys site in LTA4H, we exchanged Arg563 for a Lys, Ala, or Met, and Lys565 for an Arg, Ala, or Met, generating mutants (R563K)-, (R563A)-, (R563M)-, (K565R)-, (K565A)-, and (K565M)LTA4H. We also reversed the positions of Arg563 and Lys565, generating the double crossover mutant (R563K/K565R)LTA4H. As control, we exchanged Glu560 for a Gln, generating (E560Q)LTA4H. This control residue was selected on the basis of proximity to the active site and predicted non-catalytic nature. The resulting 8 mutants were all expressed as (His)6-tagged fusion proteins in E. coli, to allow rapid purification on nickel-nitrilotriacetic acid resins. The level of expression was similar for wild type enzyme and all mutants, with a final yield of 2.4–6.5 mg of purified protein per liter of cell culture. Catalytic Properties of Mutants of Arg563—Mutagenetic replacements of Arg563 completely eliminated the epoxide hydrolase activity of LTA4H (Table II). Thus, neither (R563K)-, (R563A)-, nor (R563M)LTA4H converted LTA4 into detectable amounts of LTB4. Although these mutations also strongly reduced the aminopeptidase activity, the effects were not quite as detrimental as for the epoxide hydrolase reaction. Thus, in (R563K)LTA4H, Vmax and Km reached 6.4 and 58%, respectively, of wild type enzyme, whereas in (R563A)LTA4H, the aminopeptidase activity was almost completely abolished, with a Vmax and Km for Ala-pNA of 0.3 and 6.2%, respectively. On the other hand, (R563M)LTA4H exhibited a Vmax and Km of 20 and 179%, respectively, of wild type enzyme (Table II). Accordingly, the specificity constants of both (R563K)- and (R563M)LTA4H were reduced to only 11% of the wild type enzyme, whereas the specificity constant for (R563A)LTA4H only reached 5%.Table IIKinetic parameters for the aminopeptidase and epoxide hydrolase activities Mutated recombinant enzymes were expressed in E. coli, purified by affinity chromatography, and assayed for the aminopeptidase and epoxide hydrolase activity. Apparent kinetic constants are expressed in % of those of the wild type enzyme. Each data point was calculated as mean ± S.E. triplicate determinations.MutantEpoxide hydrolase activityAminopeptidase activityVmaxKmkcat/KmVmaxKmkcat/Km% of WT% of WTWild type100 ± 7100 ± 25100100 ± 2100 ± 5100E560Q61 ± 360 ± 1210039 ± 4109 ± 1036R563KNDaND, not detectableND6.4 ± 0.958 ± 1511R563ANDND0.3 ± 0.016.2 ± 1.65R563MNDND20 ± 2179 ± 3711K565R95 ± 12104 ± 3392108 ± 1782 ± 21132K565A31 ± 414 ± 1621878 ± 12483 ± 11016K565M32 ± 233 ± 129558 ± 12645 ± 1739R563K/K565RNDND18 ± 347 ± 1439a ND, not detectable Open table in a new tab Catalytic Properties of Mutants of Lys565—Mutations of Lys565 had variable, but no dramatic effects on the epoxide hydrolase activity. (K565R)LTA4H exhibited a Vmax and Km of 95 and 104%, respectively, of wild type enzyme, whereas for (K565A)LTA4H, Vmax and Km for LTA4 were 31 and 14%, respectively, and for (K565M)LTA4H these values mounted to 32 and 33%, respectively, of wild type enzyme (Table II). In addition, the specificity constants for the more conservative mutant (K565R)LTA4H and for (K565M)LTA4H remained unchanged (95 and 92%, respectively) and even increased for (K565A)LTA4H (218%), indicating that the substrate binding and catalytic residues are not drastically altered in these mutants. For the aminopeptidase activity, on the other hand, exchange of Lys565 for an Ala or Met mostly affected the Michaelis constant, with only marginal effects on the Vmax value, whereas exchange of Lys565 for an Arg affected neither Vmax nor Km. Thus, for (K565A)LTA4H, Vmax and Km were 78 and 483%, respectively, of the wild type enzyme, and for (K565M)LTA4H the corresponding values were 58 and 645%, respectively. For (K565R)LTA4H, which retains the positive charge, values of Vmax and Km remained at 108 and 82%, respectively (Table II). The effects on the Michaelis constants were also reflected in the specificity constants of (K565A)- and (K565M)LTA4H, which were reduced to 16 and 9%, respectively, but increased minimally in (K565R)LTA4H to 132%. It should be noted that Lys565 is part of a salt bridge involving Glu533 and that effects of mutations may, at least to some extent, be explained by a disturbance of these charge interactions. Control Mutants (R563K/K565R)- and (E560Q)LTA4H—As expected, the double crossover mutant (R563K/K565R)LTA4H, yielded an enzyme devoid of epoxide hydrolase activity, as seen in (R563K)LTA4H. However, (R563K/K565R)LTA4H exhibited a reduced peptidase activity, exhibiting a Vmax and Km of 18 and 47%, respectively, of wild type enzyme. The specificity constant for the double crossover mutant attained 39% of that of wild type enzyme. The control mutation close to Arg563 and Lys565 did not cause any major effects on enzyme catalysis. For the epoxide hydrolase activity, (E560Q)LTA4H exhibited a Vmax and Km of 61 and 60%, respectively, of wild type enzyme (Table II). As a consequence, the specificity constant was not significantly changed. For the aminopeptidase activity, (E560Q)LTA4H exhibited a Vmax and Km of 39 and 109%, respectively, of wild type enzyme (Table II). The kcat/Km value for (E560Q)LTA4H was reduced to 36%. Analysis of LTA4 Binding Strength by Enzyme Inhibition Assays—Because all mutants in position 563 lacked epoxide hydrolase activity, inhibition assays against the residual aminopeptidase activity were carried out to probe the effects of mutations on the binding of LTA4. For (R563K)- and (R563M)LTA4H, the hydroxamic acid inhibitor exhibited a Ki of 0.1 and 0.2 μm, as compared with 0.01 μm for wild type enzyme. In contrast, no significant difference in Ki could be detected between (R563K)-, (R563M)-, and wild type LTA4H when employing LTA4 as an inhibitor of the aminopeptidase activity (Table III). Thus, the Ki for LTA4 (0–10 μm) was determined to 7.5, 5.5, and 8 μm for (R563K)-, (R563M)-, and wild type LTA4H, respectively. These values are similar to previously reported Km values for LTA4 (5–30 μm), suggesting that the substrate binds equally strong and in similar conformations to both mutated and wild type LTA4H.Table IIIInhibition assays Values for inhibition constants (Ki) are in