Saccharomyces cerevisiae leukotriene A4 hydrolase: formation of leukotriene B4 and identification of catalytic residues

Leukotriene A(4) hydrolase in mammals is a bifunctional zinc metalloenzyme that catalyzes the hydrolysis of leukotriene A(4) into the proinflammatory mediator leukotriene B(4), and also possesses an aminopeptidase activity. Recently we cloned and characterized an leukotriene A(4) hydrolase from Saccharomyces cerevisiae as a leucyl aminopeptidase with an epoxide hydrolase activity. Here we show that S. cerevisiae leukotriene A(4) hydrolase is a metalloenzyme containing one zinc atom complexed to His-340, His-344, and Glu-363. Mutagenetic analysis indicates that the aminopeptidase activity follows a general base mechanism with Glu-341 and Tyr-429 as the base and proton donor, respectively. Furthermore, the yeast enzyme hydrolyzes leukotriene A(4) into three compounds, viz., 5S,6S-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid, leukotriene B(4), and Delta(6)-trans-Delta(8)-cis-leukotriene B(4), with a relative formation of 1:0.2:0.1. In addition, exposure of S. cerevisiae leukotriene A(4) hydrolase to leukotriene A(4) selectively inactivates the epoxide hydrolase activity with a simultaneous stimulation of the aminopeptidase activity. Moreover, kinetic analyses of wild-type and mutated S. cerevisiae leukotriene A(4) hydrolase suggest that leukotriene A(4) binds in one catalytic mode and one tight-binding, regulatory mode. Exchange of a Phe-424 in S. cerevisiae leukotriene A(4) hydrolase for a Tyr, the corresponding residue in human leukotriene A(4) hydrolase, results in a protein that converts leukotriene A(4) into leukotriene B(4) with an improved efficiency and specificity. Hence, by a single point mutation, we could make the active site better suited to bind and turn over the substrate leukotriene A(4), thus mimicking a distinct step in the molecular evolution of S. cerevisiae leukotriene A(4) hydrolase toward its mammalian counterparts.     

Cloning and characterization of a bifunctional leukotriene A(4) hydrolase from Saccharomyces cerevisiae

In mammals, leukotriene A(4) hydrolase is a bifunctional zinc metalloenzyme that catalyzes hydrolysis of leukotriene A(4) into the proinflammatory leukotriene B(4) and also possesses an arginyl aminopeptidase activity. We have cloned, expressed, and characterized a protein from Saccharomyces cerevisiae that is 42% identical to human leukotriene A(4) hydrolase. The purified protein is an anion-activated leucyl aminopeptidase, as assessed by p-nitroanilide substrates, and does not hydrolyze leukotriene A(4) into detectable amounts of leukotriene B(4). However, the S. cerevisiae enzyme can utilize leukotriene A(4) as substrate to produce a compound identified as 5S,6S-dihydroxy-7,9-trans-11, 14-cis-eicosatetraenoic acid. Both catalytic activities are inhibited by 3-(4-benzyloxyphenyl)-2-(R)-amino-1-propanethiol (thioamine), a competitive inhibitor of human leukotriene A(4) hydrolase. Furthermore, the peptide cleaving activity of the S. cerevisiae enzyme was stimulated approximately 10-fold by leukotriene A(4) with kinetics indicating the presence of a lipid binding site. Nonenzymatic hydrolysis products of leukotriene A(4), leukotriene B(4), arachidonic acid, or phosphatidylcholine were without effect. Moreover, leukotriene A(4) could displace the inhibitor thioamine and restore maximal aminopeptidase activity, indicating that the leukotriene A(4) binding site is located at the active center of the enzyme. Hence, the S. cerevisiae leukotriene A(4) hydrolase is a bifunctional enzyme and appears to be an early ancestor to mammalian leukotriene A(4) hydrolases.     

Inhibition of leukotriene A4 hydrolase/aminopeptidase by captopril

Captopril ((2S)-1-(3-mercapto-2-methyl-propionyl)-L-proline) inhibited the bifunctional, Zn(2+)-containing enzyme leukotriene A4 hydrolase/aminopeptidase reversibly and competitively with Ki = 6.0 microM for leukotriene B4 formation and Ki = 60 nM for L-lysine-p-nitroanilide hydrolysis at pH 8. Inhibition was independent of pH between pH 7 and 8, the optimum range for each catalytic activity. Half-maximal inhibition of leukotriene B4 formation by intact erythrocytes and neutrophils required 50 and 88 microM captopril, respectively. In neutrophils and platelets neither 5(S)-hydroxyeicosatetraenoic acid, 12(S)-hydroxyeicosatetraenoic acid, nor leukotriene C4 formation were reduced, indicating selective inhibition of leukotriene A4 hydrolase/aminopeptidase, not 5-lipoxygenase, 12-lipoxygenase, or leukotriene C4 synthase. In whole blood, captopril inhibited leukotriene B4 formation with an accompanying redistribution of substrate toward formation of cysteinyl leukotrienes. The decrease in leukotriene B4 was more substantial than the corresponding increase in cysteinyl leukotrienes suggesting that nonenzymatic hydration predominates over transcellular metabolism of leukotriene A4 by platelets during selective inhibition of leukotriene A4 hydrolase. Enalapril dicarboxylic acid and Glu-Trp-Pro-Arg-ProGln-Ile-Pro-Pro which inhibit angiotensin-converting enzyme: angiotensin I, bradykinin, and N-[3-(2-furyl)acryloyl]Phe-Gly-Gly which are substrates; and chloride ions which activate angiotensin-converting enzyme did not modulate leukotriene A4 hydrolase/aminopeptidase activity. The results indicate that: (i) the sulfhydryl group of captopril is an important determinant for inhibition of leukotriene A4 hydrolase/aminopeptidase, probably by binding to an active site Zn2+; (ii) aminopeptidase and leukotriene A4 hydrolase display differential susceptibility to inhibition; (iii) there is minimal functional similarity between angiotensin-converting enzyme (peptidyl dipeptidase) and leukotriene A4 hydrolase/aminopeptidase; (iv) captopril may be a useful prototype to identify more potent and selective leukotriene A4 hydrolase inhibitors.     

Effect of the leukotriene A4 hydrolase aminopeptidase augmentor 4-methoxydiphenylmethane in a pre-clinical model of pulmonary emphysema

The leukotriene A(4) hydrolase enzyme is a dual functioning enzyme with the following two catalytic activities: an epoxide hydrolase function that transforms the lipid metabolite leukotriene A(4) to leukotriene B(4) and an aminopeptidase function that hydrolyzes short peptides. To date, all drug discovery efforts have focused on the epoxide hydrolase activity of the enzyme, because of extensive biological characterization of the pro-inflammatory properties of its metabolite, leukotriene B(4). Herein, we have designed a small molecule, 4-methoxydiphenylmethane, as a pharmacological agent that is bioavailable and augments the aminopeptidase activity of the leukotriene A(4) hydrolase enzyme. Pre-clinical evaluation of our drug showed protection against intranasal elastase-induced pulmonary emphysema in murine models.     

Leukotriene A4 hydrolase: an epoxide hydrolase with peptidase activity

Purified leukotriene A4 hydrolase from human leukocytes is shown to exhibit peptidase activity towards the synthetic substrates alanine-4-nitroanilide and leucine-4-nitroanilide. The enzymatic activity is abolished after heat treatment (70 degrees C, 30 min). At 37 degrees C these substrates are hydrolyzed at a rate of 380 and 130 nmol/mg/min, respectively, and there is no enzyme inhibition during catalysis. Apo-leukotriene A4 hydrolase, obtained by removal of the intrinsic zinc atom, exhibits only a low peptidase activity which can be restored by the addition of stoichiometric amounts of zinc. Reconstitution of the apoenzyme with cobalt results in a peptidase activity which exceeds that of enzyme reactivated with zinc. Preincubation of the native enzyme with leukotriene A4 reduces the peptidase activity. Semipurified preparations of bovine intestinal aminopeptidase and porcine kidney aminopeptidase do not hydrolyze leukotriene A4 into leukotriene B4.     

Leukotriene A4 hydrolase, a bifunctional enzyme. Distinction of leukotriene A4 hydrolase and aminopeptidase activities by site-directed mutagenesis at Glu-297.

We previously obtained evidence for intrinsic aminopeptidase activity for leukotriene (LT)A4 hydrolase, an enzyme characterized to specifically catalyse the hydrolysis of LTA4 to LTB4, a chemotactic compound. From a sequence homology search between LTA4 hydrolase and several aminopeptidases, it became clear that they share a putative active site for known aminopeptidases and a zinc binding domain. Thus, Glu-297 of LTA4 hydrolase is a candidate for the active site of its aminopeptidase activity, while His-296, His-300 and Glu-319 appear to constitute a zinc binding site. To determine whether or not this putative active site is also essential to LTA4 hydrolase activity, site-directed mutagenesis experiments were carried out. Glu-297 was mutated into 4 different amino acids. The mutant E297Q (Glu changed to Gln) conserved LTA4 hydrolase activity but showed little aminopeptidase activity. Other mutants at Glu-297 (E297A, E297D and E297K) showed markedly reduced amounts of both activities. It is thus proposed that either a glutamic or glutamine moiety at 297 is required for full LTA4 hydrolase activity, while the free carboxylic acid of glutamic acid is essential for aminopeptidase.     

Leukotriene A4 hydrolase is a zinc-containing aminopeptidase

A comparison of amino acid sequences revealed that leukotriene A4 (LTA4) hydrolase is homologous to various types of aminopeptidases. Consistently with the finding, the purified LTA4 hydrolases from both human and guinea pig sources contained equimolar zinc ion, as determined by atomic absorption spectrometry. The enzyme had a significant amount of aminopeptidase activity toward synthetic peptide substrates. Both LTA4 hydrolase and aminopeptidase activities were inhibited by o-phenanthroline, p-chloromercuribenzoic acid, and Leu-thiol with similar IC50 values. Co-purification as well as co-immunoprecipitation of both enzyme activities with an affinity-purified antibody against LTA4 hydrolase strongly suggest that the two enzyme activities reside in a single protein.     

Nuclear localization of leukotriene A4 hydrolase in type II alveolar epithelial cells in normal and fibrotic lung

Leukotriene A4 (LTA4) hydrolase catalyzes the final step in leukotriene B4 (LTB4) synthesis. In addition to its role in LTB4 synthesis, the enzyme possesses aminopeptidase activity. In this study, we sought to define the subcellular distribution of LTA4 hydrolase in alveolar epithelial cells, which lack 5-lipoxygenase and do not synthesize LTA4. Immunohistochemical staining localized LTA4 hydrolase in the nucleus of type II but not type I alveolar epithelial cells of normal mouse, human, and rat lungs. Nuclear localization of LTA4 hydrolase was also demonstrated in proliferating type II-like A549 cells. The apparent redistribution of LTA4 hydrolase from the nucleus to the cytoplasm during type II-to-type I cell differentiation in vivo was recapitulated in vitro. Surprisingly, this change in localization of LTA4 hydrolase did not affect the capacity of isolated cells to convert LTA4 to LTB4. However, proliferation of A549 cells was inhibited by the aminopeptidase inhibitor bestatin. Nuclear accumulation of LTA4 hydrolase was also conspicuous in epithelial cells during alveolar repair following bleomycin-induced acute lung injury in mice, as well as in hyperplastic type II cells associated with fibrotic lung tissues from patients with idiopathic pulmonary fibrosis. These results show for the first time that LTA4 hydrolase can be accumulated in the nucleus of type II alveolar epithelial cells and that redistribution of the enzyme to the cytoplasm occurs with differentiation to the type I phenotype. Furthermore, the aminopeptidase activity of LTA4 hydrolase within the nucleus may play a role in promoting epithelial cell growth.     

Amino-benzosuberone: a novel warhead for selective inhibition of human aminopeptidase-N/CD13

This paper describes the design and synthesis of compounds belonging to a novel class of highly selective mammalian CD13 inhibitors. Racemic homologues of 3-amino-2-tetralone 1 were synthesised and evaluated for their ability to selectively inhibit the membrane-bound, zinc-dependent aminopeptidase-N/CD13 (EC 3.4.11.2). Some of these novel non-peptidic compounds are potent, competitive inhibitors of the mammalian enzyme, with K(i) values in the low micromolar range in spite of their minimal size (MW <200 Da). Moreover, they show an interesting selectivity profile against representative members of the aminopeptidase family, that is leucine aminopeptidase (EC 3.4.11.1), Aeromonas proteolytica aminopeptidase (EC 3.4.11.10) and the aminopeptidase activity of leukotriene A4 hydrolase (EC 3.3.2.6). The amino-benzosuberone derivative 4 is the most promising compound in terms of potency, stability and selectivity. A hypothetical binding mode of 4 to the catalytic zinc and several conserved active site residues is proposed, based on the observed structure-activity relationships, structural insights from aminopeptidase-N homologues of known three-dimensional structure.     

Leukotriene A4 hydrolase, insights into the molecular evolution by homology modeling and mutational analysis of enzyme from Saccharomyces cerevisiae

Mammalian leukotriene A4 (LTA4) hydrolase is a bifunctional zinc metalloenzyme possessing an Arg/Ala aminopeptidase and an epoxide hydrolase activity, which converts LTA4 into the chemoattractant LTB4. We have previously cloned an LTA4 hydrolase from Saccharomyces cerevisiae with a primitive epoxide hydrolase activity and a Leu aminopeptidase activity, which is stimulated by LTA4. Here we used a modeled structure of S. cerevisiae LTA4 hydrolase, mutational analysis, and binding studies to show that Glu-316 and Arg-627 are critical for catalysis, allowing us to a propose a mechanism for the epoxide hydrolase activity. Guided by the structure, we engineered S. cerevisiae LTA4 hydrolase to attain catalytic properties resembling those of human LTA4 hydrolase. Thus, six consecutive point mutations gradually introduced a novel Arg aminopeptidase activity and caused the specific Ala and Pro aminopeptidase activities to increase 24 and 63 times, respectively. In contrast to the wild type enzyme, the hexuple mutant was inhibited by LTA4 for all tested substrates and to the same extent as for the human enzyme. In addition, these mutations improved binding of LTA4 and increased the relative formation of LTB4, whereas the turnover of this substrate was only weakly affected. Our results suggest that during evolution, the active site of an ancestral eukaryotic zinc aminopeptidase has been reshaped to accommodate lipid substrates while using already existing catalytic residues for a novel, gradually evolving, epoxide hydrolase activity. Moreover, the unique ability to catalyze LTB4 synthesis appears to be the result of multiple and subtle structural rearrangements at the catalytic center rather than a limited set of specific amino acid substitutions.     

Leukotriene A4 hydrolase. Inhibition by bestatin and intrinsic aminopeptidase activity establish its functional resemblance to metallohydrolase enzymes.

Bestatin, an inhibitor of aminopeptidases, was also a potent inhibitor of leukotriene (LT) A4 hydrolase. On isolated enzyme its effects were immediate and reversible with a Ki = 201 +/- 95 mM. With erythrocytes it inhibited LTB4 formation greater than 90% within 10 min; with neutrophils it inhibited LTB4 formation by only 10% during the same period, increasing to 40% in 2 h. Bestatin inhibited LTA4 hydrolase selectively; neither 5-lipoxygenase nor 15-lipoxygenase activity in neutrophil lysates was affected. Purified LTA4 hydrolase exhibited an intrinsic aminopeptidase activity, hydrolyzing L-lysine-p-nitroanilide and L-leucine-beta-naphthylamide with apparent Km = 156 microM and 70 microM and Vmax = 50 and 215 nmol/min/mg, respectively. Both LTA4 and bestatin suppressed the intrinsic aminopeptidase activity of LTA4 hydrolase with apparent Ki values of 5.3 microM and 172 nM, respectively. Other metallohydrolase inhibitors tested did not reduce LTA4 hydrolase/aminopeptidase activity, with one exception; captopril, an inhibitor of angiotensin-converting enzyme, was as effective as bestatin. The results demonstrate a functional resemblance between LTA4 hydrolase and certain metallohydrolases, consistent with a molecular resemblance at their putative Zn2(+)-binding sites. The availability of a reversible, chemically stable inhibitor of LTA4 hydrolase may facilitate investigations on the role of LTB4 in inflammation, particularly the process termed transcellular biosynthesis.     

Synthesis and structure activity relationships of novel non-peptidic metallo-aminopeptidase inhibitors

Racemic derivatives of 3-amino-2-tetralone were synthesised and evaluated for their ability to inhibit metallo-aminopeptidase activities. New compounds substituted in position 2 by methyl ketone, substituted oximes or hydroxamic acids as well as heterocyclic derivatives were evaluated against representative members of zinc-dependent aminopeptidases: leucine aminopeptidase (E.C. 3.4.11.1), aminopeptidase-N (E.C. 3.4.11.2), Aeromonas proteolytica aminopeptidase (E.C. 3.4.11.10), and the aminopeptidase activity of leukotriene A(4) hydrolase (E.C. 3.3.2.6). Several compounds showed K(i) values in the low micromolar range against the 'one-zinc' aminopeptidases, while most of them were rather poor inhibitors of the 'two-zinc' enzymes. This interesting selectivity profile may guide the design of new, specific inhibitors of target mammalian aminopeptidases with one active site zinc.     

A conserved tyrosine residue of Saccharomyces cerevisiae leukotriene A4 hydrolase stabilizes the transition state of the peptidase activity

Saccharomyces cerevisiae leukotriene A4 hydrolase (LTA4H) is a bifunctional aminopeptidase/epoxide hydrolase and a member of the M1 family of metallopeptidases. In order to obtain a more thorough understanding of the aminopeptidase activity of the enzyme, two conserved tyrosine residues, Tyr244 and Tyr456, were altered to phenylalanine and the mutant proteins characterized by determining KM and kcat for various amino acid beta-naphthylamide substrates. While mutation of Tyr456 exhibited minimal effect on catalysis, mutation of Tyr244 caused an overall 25-100-fold reduction in catalytic activity for all substrates tested. Furthermore, LTA4H Y244F exhibited a 40-fold decrease in affinity for RB-3014, a transition state analog inhibitor, implicating Tyr244 in transition state stabilization.     

Leukotriene A4 hydrolase/aminopeptidase. Glutamate 271 is a catalytic residue with specific roles in two distinct enzyme mechanisms.

Leukotriene A(4) hydrolase/aminopeptidase is a bifunctional zinc metalloenzyme that converts the fatty acid epoxide leukotriene A(4) into leukotriene B(4), a potent chemoattractant and immune-modulating lipid mediator. Recently, the structure of leukotriene A(4) hydrolase revealed that Glu-271, which belongs to a conserved GXMEN motif in the M1 family of zinc peptidases, and Gln-136 are located at the active site. Here we report that mutagenetic replacements of Glu-271, but not Gln-136, abrogate both catalytic activities of leukotriene A(4) hydrolase. Furthermore, the 2.1 A crystal structure of [E271Q]leukotriene A(4) hydrolase revealed minimal conformational changes that could not explain the loss of enzyme function. We propose that the carboxylate of Glu-271 participates in an acid-induced opening of the epoxide moiety of leukotriene A(4) and formation of a carbocation intermediate. Moreover, Glu-271 appears to act as an N-terminal recognition site and may potentially stabilize the transition-state during turnover of peptides, a property that most likely pertains to all members of the M1 family of zinc aminopeptidases. Hence, Glu-271 is a unique example of an amino acid, which has dual and separate functions in two different catalytic reactions, involving lipid and peptide substrates, respectively.     

Molecular dynamics simulation study and hybrid pharmacophore model development in human LTA4H inhibitor design

Human leukotriene A4 hydrolase (hLTA4H) is a bi-functional enzyme catalyzes the hydrolase and aminopeptidase functions upon the fatty acid and peptide substrates, respectively, utilizing the same but overlapping binding site. Particularly the hydrolase function of this enzyme catalyzes the rate-limiting step of the leukotriene (LT) cascade that converts the LTA4 to LTB4. This product is a potent pro-inflammatory activator of inflammatory responses and thus blocking this conversion provides a valuable means to design anti-inflammatory agents. Four structurally very similar chemical compounds with highly different inhibitory profile towards the hydrolase function of hLTA4H were selected from the literature. Molecular dynamics (MD) simulations of the complexes of hLTA4H with these inhibitors were performed and the results have provided valuable information explaining the reasons for the differences in their biological activities. Binding mode analysis revealed that the additional thiophene moiety of most active inhibitor helps the pyrrolidine moiety to interact the most important R563 and K565 residues. The hLTA4H complexes with the most active compound and substrate were utilized in the development of hybrid pharmacophore models. These developed pharmacophore models were used in screening chemical databases in order to identify lead candidates to design potent hLTA4H inhibitors. Final evaluation based on molecular docking and electronic parameters has identified three compounds of diverse chemical scaffolds as potential leads to be used in novel and potent hLTA4H inhibitor design.     

Contribution of molecular modeling and site-directed mutagenesis to the identification of two structural residues,Arg-220 and Asp-227, in aminopeptidase A

Aminopeptidase A is a zinc metalloenzyme involved in the formation of brain angiotensin III, which exerts a tonic stimulatory action on the central control of blood pressure. Thus, central inhibitors of aminopeptidase A constitute putative central antihypertensive agents. Mutagenic studies have been performed to investigate organization of the aminopeptidase A active site, with a view to designing such inhibitors. The structure of one monozinc aminopeptidase (leukotriene A(4) hydrolase) was recently resolved and used to construct a three-dimensional model of the aminopeptidase A ectodomain. This new model, highly consistent with the results of mutagenic studies, showed a critical structural interaction between two conserved residues, Arg-220 and Asp-227. Mutagenic replacement of either of these two residues disrupted maturation and subcellular localization and abolished the enzymatic activity of aminopeptidase A, confirming the critical structural role of these residues. In this study, we generated the first three-dimensional model of a strict aminopeptidase, aminopeptidase A. This model constitutes a new tool to probe further the active site of aminopeptidase A and to design new inhibitors of this enzyme.     

Alteration of human leukotriene A4 hydrolase activity after site-directed mutagenesis: serine-415 is a regulatory residue.

Leukotriene A4 hydrolase (LTA-H) is a bifunctional protein that has aminopeptidase activity, but also contains an epoxide hydrolase activity that converts leukotriene (LT)A4 to LTB4. The lipid metabolic activity of this enzyme plays a central role in the control of polymorphonuclear leukocyte function and in the development of inflammation. LTA-H is widely spread in many mammalian tissues, although it appears to be inactive in many cases. Regulation of this enzyme's activity by phosphorylation of a serine at residue 415 has recently been described. Since the activation of LTA-H in the presence of activated PMNL would likely lead to a substantial increase in the production of inflammatory lipids, regulation of LTA-H presents a novel potential target for anti-inflammatory therapy. We have now made a series of site-directed mutants at this site to test the importance of this residue to the activity of LTA-H. Replacement of the critical serine with threonine or glutamine has little effect on either the epoxide hydrolase or aminopeptidase activities. However, replacing serine with a negatively charged amino acid (either aspartate or glutamate), intended to mimic phosphorylation at that site, causes significant reduction in epoxide hydrolase activity (50-70%). These mutations have little effect on the aminopeptidase activity of the LTA-H, suggesting that the mutation models the regulatory event and is not simply due to improper folding of the protein.     

Impairment of IGF-I gene splicing and MGF expression associated with muscle wasting

An aminopeptidase was purified from bovine skeletal muscle by ammonium sulfate fractionation and by successive chromatographies of DEAE-cellulose, Sehacryl S-200, phenyl-sepharose CL-4B, hydroxyapatite and Hi-Trap chelating HP columns. The aminopeptidase was purified about 14-fold over the crude extract with a yield of 1.0% activity. The molecular mass of the enzyme was found to be 58 kDa on SDS-PAGE. The enzyme activity was enhanced by the addition of some anions, such as Cl(-), NO(3)(-) and SCN(-), which is the most unique property of this enzyme. While, the activity was strongly inhibited by bestatin, PMSF and puromycin, suggesting that it was a serine protease. In addition, this enzyme was identical with leukotriene (LT) A4 hydrolase, converting LTA4 to LTB4.     

Alteration of human leukotriene A4 hydrolase activity after site-directed mutagenesis: serine-415 is a regulatory residue

Leukotriene A₄ hydrolase (LTA-H) is a bifunctional protein that has aminopeptidase activity, but also contains an epoxide hydrolase activity that converts leukotriene (LT)A₄ to LTB₄. The lipid metabolic activity of this enzyme plays a central role in the control of polymorphonuclear leukocyte function and in the development of inflammation. LTA-H is widely spread in many mammalian tissues, although it appears to be inactive in many cases. Regulation of this enzyme’s activity by phosphorylation of a serine at residue 415 has recently been described. Since the activation of LTA-H in the presence of activated PMNL would likely lead to a substantial increase in the production of inflammatory lipids, regulation of LTA-H presents a novel potential target for anti-inflammatory therapy. We have now made a series of site-directed mutants at this site to test the importance of this residue to the activity of LTA-H. Replacement of the critical serine with threonine or glutamine has little effect on either the epoxide hydrolase or aminopeptidase activities. However, replacing serine with a negatively charged amino acid (either aspartate or glutamate), intended to mimic phosphorylation at that site, causes significant reduction in epoxide hydrolase activity (50–70%). These mutations have little effect on the aminopeptidase activity of the LTA-H, suggesting that the mutation models the regulatory event and is not simply due to improper folding of the protein.     

Purification and characterisation of leukotriene A4 hydrolase from rat neutrophils

Leukotriene A4 hydrolase was rapidly and extensively purified from rat neutrophils using anion exchange and gel filtration high-pressure liquid chromatography. The enzyme which converts the allylic epoxide leukotriene A4 to the 5,12-dihydroxyeicosatetraenoic acid leukotriene B4 was localized in the cytosolic fraction and exhibited an optimum activity at pH 7.8 and an apparent Km for leukotriene A4 between 2 X 10(-5) and 3 X 10(-5) M. The purified leukotriene A4 hydrolase was shown to have a molecular weight of 68 000 on sodium dodecylsulfate polyacrylamide gel electrophoresis and of 50 000 by gel filtration. The molecular weight and monomeric native form of this enzyme are unique characteristics which distinguish leukotriene A4 hydrolase from previously purified epoxide hydrolases.