Leukotriene A5 is a substrate and an inhibitor of rat and human neutrophil LTA4 hydrolase

The epoxide 5(S) trans-5,6 oxido, 7,9 trans-11,14,17 cis eicosatetraenoic acid (leukotriene A5) was chemically synthesized and demonstrated to be both a substrate and an inhibitor of partially purified rat and human LTA4 hydrolase. Both rat and human LTA4 hydrolase utilized leukotriene A5 less effectively as a substrate than leukotriene A4. Incubation of leukotriene A5 (10 microM) or leukotriene A4 (10 microM) with rat neutrophils demonstrated formation of 123 pmol LTB5/min/10(7) cells and 408 pmol LTB4/min/10(7) cells respectively. Purified rat neutrophil LTA4 hydrolase incubated with 100 microM leukotriene A5 produced 22 nmol LTB5/min/mg protein and when incubated with 100 microM leukotriene A4 produced 50 nmol LTB4/min/mg protein. Human neutrophil LTA4 hydrolase incubated with 100 microM leukotriene A5 produced 24 nmol LTB5/min/mg protein and when incubated with 100 microM leukotriene A4 produced 52 nmol LTB4/min/mg protein. Leukotriene A5 was an inhibitor of the formation of leukotriene B4 from leukotriene A4 by both the rat and human neutrophil LTA4 hydrolase. Excess leukotriene A5 prevented covalent coupling of [3H] leukotriene A4 to LTA4 hydrolase suggesting inhibition may involve covalent coupling of leukotriene A5 to the LTA4 hydrolase.     

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.     

Purification and characterization of leukotriene A4 epoxide hydrolase from dog lung

Leukotriene A₄ epoxide hydrolase from dog lung, a soluble enzyme catalyzing the hydrolysis of leukotriene A₄ (LTA₄) to leukotriene B₄ (LTB₄) was partially purified by anion exchange HPLC. The enzymatic reaction obeys Michaelis- Menten kinetics. The apparent Km ranged between 15 and 25 μM and the enzyme exhibited an optimum activity at pH 7.8. An improved assay for the epoxide hydrolase has been developed using bovine serum albumin and EDTA to increase the conversion of LTA₄ to LTB₄. This method was used to produce 700 mg of LTB₄ from LTA₄ methyl ester. The partial by purified enzyme was found to be uncompetitively inhibited by divalent cations. Ca²⁺, Mn⁺, Fe²⁺, Zn⁺² and Cu⁺² were found to have inhibitor constants (Ki) of 89 mM, 3.4 mM, 1.1 mM, 0.57 mM, and 28 μM respectively Eicosapentaenoic acid was shown to be a competitive inhibitor of this enzyme with a Ki of 200 μM. From these inhibition studies, it can be theorized that the epoxide hydrolae has at least one hydrophobic and one hydrophilic binding site.     

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 A3. A poor substrate but a potent inhibitor of rat and human neutrophil leukotriene A4 hydrolase

Analysis of leukotriene B4 production by purified rat and human neutrophil leukotriene (LT) A4 hydrolases in the presence of 5(S)-trans-5,6-oxido-7,9-trans-11-cis-eicosatrienoic acid (leukotriene A3) demonstrated that this epoxide is a potent inhibitor of LTA4 hydrolase. Insignificant amounts of 5(S), 12(R)-dihydroxy-6-cis-8,10-trans-eicosatrienoic acid (leukotriene B3) were formed by incubation of rat neutrophils with leukotriene A3 or by the purified rat and human LTA4 hydrolases incubated with leukotriene A3. Leukotriene A3 was shown to be a potent inhibitor of leukotriene B4 production by rat neutrophils and also by purified rat and human LTA4 hydrolases. Covalent coupling of [3H]leukotriene A4 to both rat and human neutrophil LTA4 hydrolases was shown, and this coupling was inhibited by preincubation of the enzymes with leukotriene A4. Preincubation of rat neutrophils with leukotriene A3 also prevented labeling of LTA4 hydrolase by [3H]leukotriene A4. This result indicates that leukotriene A3 prevents covalent coupling of the substrate leukotriene A4 and inhibits the production of leukotriene B4 by blocking the binding of leukotriene A4 to the enzyme.     

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.     

Solubilization and partial purification of leukotriene C4 synthase from guinea-pig lung: a microsomal enzyme with high specificity towards 5,6-epoxide leukotriene A4

Enzymic activities catalyzing allylic epoxide, leukotriene A4, to leukotriene C4 by conjugation with glutathione were present mainly in microsomal fractions of spleens and lungs of guinea pigs and rats. Leukotriene C4 (LTC4) synthase was solubilized from the microsomes of guinea-pig lung by the new procedures of a combination of 3-[3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS), digitonin and KCl. The enzyme was partially purified by two steps of column chromatography which resulted in a complete resolution of the enzyme from glutathione S-transferases (EC 2.5.1.18). The partially purified LTC4 synthase showed a Vmax value of 40 nmol/min per mg, and the apparent Km values for LTA4 and glutathione were 36 microM and 1.6 mM, respectively. The enzyme was unstable, and half of the activity was lost by incubation at 37 degrees C for 3 min. Glutathione at 10 mM completely protected the enzyme against this inactivation, while other sulfhydryl-group-reducing reagents were ineffective. The partially purified enzyme revealed a high specificity towards 5,6-epoxide leukotrienes (LTA4 and its methyl ester), while rat cytosolic glutathione S-transferases catalyzed conjugation of glutathione to various positional isomers of epoxide leukotrienes.     

Development of a homogeneous time-resolved fluorescence leukotriene B4 assay for determining the activity of leukotriene A4 hydrolase

Leukotriene A4 (LTA4) hydrolase catalyzes a rate-limiting final biosynthetic step of leukotriene B4 (LTB4), a potent lipid chemotactic agent and proinflammatory mediator. LTB4 has been implicated in the pathogenesis of various acute and chronic inflammatory diseases, and thus LTA4 hydrolase is regarded as an attractive therapeutic target for anti-inflammation. To facilitate identification and optimization of LTA4 hydrolase inhibitors, a specific and efficient assay to quantify LTB4 is essential. This article describes the development of a novel 384-well homogeneous time-resolved fluorescence assay for LTB4 (LTB4 HTRF assay) and its application to establish an HTRF-based LTA4 hydrolase assay for lead optimization. This LTB4 HTRF assay is based on competitive inhibition and was established by optimizing the reagent concentration, buffer composition, incubation time, and assay miniaturization. The optimized assay is sensitive, selective, and robust, with a Z' factor of 0.89 and a subnanomolar detection limit for LTB4. By coupling this LTB4 HTRF assay to the LTA4 hydrolase reaction, an HTRF-based LTA4 hydrolase assay was established and validated. Using a test set of 16 LTA4 hydrolase inhibitors, a good correlation was found between the IC50 values obtained using LTB4 HTRF with those determined using the LTB enzyme-linked immunoassay (R = 0.84). The HTRF-based LTA4 hydrolase assay was shown to be an efficient and suitable assay for determining compound potency and library screening to guide the development of potent inhibitors of LTA4 hydrolase.     

Crystal structure of human leukotriene A(4) hydrolase, a bifunctional enzyme in inflammation

Leukotriene (LT) A(4) hydrolase/aminopeptidase (LTA4H) is a bifunctional zinc enzyme that catalyzes the biosynthesis of LTB4, a potent lipid chemoattractant involved in inflammation, immune responses, host defense against infection, and PAF-induced shock. The high resolution crystal structure of LTA4H in complex with the competitive inhibitor bestatin reveals a protein folded into three domains that together create a deep cleft harboring the catalytic Zn(2+) site. A bent and narrow pocket, shaped to accommodate the substrate LTA(4), constitutes a highly confined binding region that can be targeted in the design of specific anti-inflammatory agents. Moreover, the structure of the catalytic domain is very similar to that of thermolysin and provides detailed insight into mechanisms of catalysis, in particular the chemical strategy for the unique epoxide hydrolase reaction that generates LTB(4).     

Leukotriene A4: preparation and enzymatic conversion in a cell-free system to leukotriene B4

Treatment of leukotriene A4 (LTA4) methyl ester with sodium hydroxide in aqueous methanol at 4 degrees C afforded LTA4, the presence of which was inferred from the UV spectrum of the compound, its rate of reaction with water, and the identity of the hydration products obtained. The half-life of LTA4 in water (pH 7.4, room temperature) was increased from 14 to 500 s by 1 mg/ml of bovine serum albumin. This stabilized (chiral) LTA4 was converted to LTB4 by an epoxide hydrolase activity in the 100,000 x g supernatant fraction from sonified rat basophilic leukemia cells. Neither the ester of LTA4 nor the biologically incorrect enantiomer of LTA4 was metabolized to LTB4 under these conditions.     

Modulation of human neutrophil LTA hydrolase activity by phorbol myristate acetate

The phorbol ester, phorbol 12-myristate 13-acetate enhanced leukotriene B4 production stimulated by formyl-methionyl-leucyl-phenylalanine and arachidonic acid and reduced the production of the all-trans isomers of LTB4 by human neutrophils. Production of 5-hydroxyeicosatetraenoic acid was unaffected. These observations are consistent with a stimulatory effect of phorbol ester on LTA hydrolase, the enzyme which catalyses the conversion of LTA4 to LTB4. We demonstrate that a protein of the same molecular weight as LTA hydrolase is phosphorylated upon stimulation of neutrophils with PMA. These data suggest that the activity of LTA hydrolase may be regulated by protein kinase C-dependent phosphorylation.     

Involvement of leukotriene B4 in arthritis models

We investigated the role of leukotriene B4 (LTB4) in murine arthritis models using a leukotriene A4 (LTA4) hydrolase inhibitor, SA6541. SA6541 inhibited the severity of collagen-induced arthritis and muramyl dipeptide (MDP)-induced hyperproliferation of synovial cells in vivo. SA6541 also inhibited LTA4-induced hyperproliferation of synovial stromal cells in vitro. These results suggest that LTB4 may play an important role in arthritis models.     

Expression and purification of rat recombinant aminopeptidase B secreted from baculovirus-infected insect cells

Aminopeptidase B (Ap-B) is a ubiquitous enzyme and its physiological function still remains an open question. This Zn2+ -exopeptidase catalyzes the amino-terminal cleavage of basic residues of peptide or protein substrates, indicating a role in precursor processing. In addition, the enzyme exhibits a residual capacity to hydrolyze leukotriene A4 (LTA4) into the pro-inflammatory lipid mediator leukotriene B4 (LTB4) in vitro. This potential bi-functional nature of Ap-B is supported by a close structural relationship with LTA4 hydrolase, which hydrolyzes LTA4 into LTB4, in vivo, and exhibits an aminopeptidase activity, in vitro. Structural studies are necessary for the detailed understanding of the bi-functional enzymatic mechanism of Ap-B. In this study, we report cDNA cloning, baculovirus expression, and purification of the rat Ap-B (rAp-B). The Ap-B cDNA was constructed from extracted rat testes total RNA and introduced into the pBAC1 baculovirus transfer vector to generate recombinant baculoviruses. rAp-B expression, with or without COOH-hexahistidine tag, was tested in two different insect cell hosts (Sf9 and H5). The enzyme is secreted into the insect cell culture medium, which allowed a rapid purification of the protein. The His-tagged rAp-B was purified using metal affinity resin while the native recombinant rAp-B was partially purified using a single step DEAE Trisacryl ion exchange column. Although the recombinant rAp-B exhibits biochemical properties equivalent to those of the rat testes purified protein, the presence of the histidine-tag seems to partially inhibit the exopeptidase activity. However, this report shows that baculovirus-infected cells are a useful system to produce rat Ap-B for use in studying enzymatic mechanisms in vitro and 3D structure.     

Leukotriene A4 hydrolase in human leukocytes. Purification and properties

Leukotriene A4 hydrolase, a soluble enzyme catalyzing hydrolysis of the allylic epoxide leukotriene A4 to the dihydroxy acid leukotriene B4, was purified to apparent homogeneity from human leukocytes. The enzymatic reaction obeyed Michaelis-Menten saturation kinetics with respect to varying concentrations of leukotriene A4. An apparent KM value ranging between 20 and 30 microM was deduced from Eadie-Hofstee plots. Physical properties including molecular weight (68,000-70,000), amino acid composition, and aminoterminal sequence were determined. It was indicated that leukotriene A4 hydrolase is a monomeric protein, distinct from previously described epoxide hydrolases in liver.     

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.     

Purification and properties of inositol-1,4-bisphosphate 4-phosphohydrolase from rat brain

Inositol-1,4-bisphosphate 4-phosphohydrolase (inositol-1,4-bisphosphatase) was highly purified from a soluble fraction of rat brain. On SDS-polyacrylamide gel electrophoresis, the purified enzyme gave a single protein band and its molecular weight was estimated to be 42000. The isoelectric point of the enzyme was 4.3. The enzyme specifically hydrolyzed the 4-phosphomonoester linkage of inositol 1,4-bisphosphate. The Km value for inositol 1,4-bisphosphate was 30 microM, and it required Mg2+ for activity. Ca2+ was a competitive inhibitor with a Ki value of 60 microM as regards the Mg2+ binding. Li+, which is known to be a strong inhibitor of inositol 1-phosphatase (EC 3.1.3.25), inhibited the enzyme activity and caused 50% inhibition at a concentration of 1 mM (IC50 = 1 mM). Li+ was an uncompetitive inhibitor of substrate binding with a Ki value of 0.6 mM. These inhibitory parameters of Li+ were quite similar to those for inositol 1-phosphatase (IC50 = 1 mM, Ki = 0.3 mM). Thus, the effect of Li+ on decreasing the free inositol level with a subsequent decrease in agonist-sensitive phosphoinositides, is caused by its inhibition of multiple enzymes involved in conversion of inositol 1,4-bisphosphate to inositol.     

Guinea-pig liver leukotriene A4 hydrolase. Purification, characterization and structural properties

Leukotriene A4 hydrolase from perfused guinea-pig liver was purified 1200-fold to near homogeneity with a yield of about 20%. Apparent values of Km and Vmax at 37 degrees C (27 microM and 68 mumol x mg-1 x min-1), turnover number, and activation energy for the conversion of leukotriene A4 into leukotriene B4 were estimated from kinetic data obtained at -10 degrees C, 0 degree C and +10 degrees C (Arrhenius plots). Physical properties including Mr (67,000-71,000), pH optimum, isoelectric point and Stokes' radius were determined. The amino acid composition and N-terminal amino acid sequence were established after carboxymethylation of the enzyme. Unlike liver cytosolic epoxide hydrolase, the purified enzyme did not catalyze the conversion of leukotriene A4 into (5S,6R)-5,6-dihydroxy-7,9-trans-11,14-cis-icosatetraenoic acid.     

Enzymatic hydration of leukotriene A4. Purification and characterization of a novel epoxide hydrolase from human erythrocytes

Human erythrocytes contained a soluble cytosolic epoxide hydrolase for stereospecific enzymatic hydration of leukotriene A4 into leukotriene B4. The enzyme was purified 1100-fold, to apparent electrophoretic homogeneity, by conventional DEAE-Sephacel fractionation followed by high performance anion exchange and chromatofocusing procedures. Its characteristics include a molecular weight of 54,000 +/- 1,000, an isoelectric point 4.9 +/- 0.2, a Km apparent from 7 to 36 microM for enzymatic hydration of leukotriene A4, and a pH optimum ranging from 7 to 8. The enzyme was partially inactivated by its initial exposure to leukotriene A4. There was slow but detectable enzymatic hydration (pmol/min/mg) of certain arachidonic acid epoxides including (+/-)-14,15-oxido-5,8-11-eicosatrienoic acid and (+/-)-11,12-oxido-5,8,14-eicosatrienoic acid, but not others, including 5,6-oxido-8,11,14-eicosatrienoic acid. Human erythrocyte epoxide hydrolase did not hydrate either styrene oxide or trans-stilbene oxide. In terms of its physical properties and substrate preference for leukotriene A4, the erythrocyte enzyme differs from previously described versions of epoxide hydrolase. Human erythrocytes represent a novel source for an extrahepatic, cytosolic epoxide hydrolase with a potential physiological role.     

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.     

Opioid peptides are substrates for the bifunctional enzyme LTA4 hydrolase/aminopeptidase.

We determined if any naturally occurring peptides could act as substrates or inhibitors of the bifunctional, Zn2+ metalloenzyme LTA4 hydrolase/aminopeptidase (E.C.3.3.2.6). Several opioid peptides including met5-enkephalin, leu5-enkephalin, dynorphin1-6, dynorphin1-7, and dynorphin1-8 competitively inhibited the hydrolysis of L-proline-p-nitroanilide by leukotriene A4 hydrolase/aminopeptidase, consistent with an interaction at its active site. The enzyme catalyzed the N-terminal hydrolysis of tyrosine from met5-enkephalin with Km = 450 +/- 58 microM and Vmax = 4.9 +/- 0.6 nmol-hr-1-ug-1 and from leu5-enkephalin with Km = 387 +/- 90 microM and Vmax = 6.2 +/- 2.5 nmol-hr-1-ug-1. Bestatin, captopril and carnosine inhibited the hydrolysis of the enkephalins. It is noteworthy that the bifunctional catalytic traits of this enzyme include generation of an hyperalgesic substance, LTB4, and inactivation of analgesic opioid peptides.