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Jesper Z. Haeggström - One of the best experts on this subject based on the ideXlab platform.
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Structure and catalytic mechanisms of Leukotriene A4 hydrolase.
Prostaglandins & Other Lipid Mediators, 2007Co-Authors: Jesper Z. Haeggström, Fredrik Tholander, Anders WetterholmAbstract:Abstract Leukotriene A4 hydrolase catalyzes the final and committed step in the biosynthesis of Leukotriene B4, a potent chemotactic agent for neutrophils, eosinophils, monocytes, and T-cells that play key roles in the innate immune response. Recent data strongly implicates Leukotriene B4 in the pathogenesis of cardiovascular diseases, in particular arteriosclerosis, myocardial infarction and stroke. Here, we highlight the most salient features of Leukotriene A4 hydrolase with emphasis on its biochemistry and structure biology.
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80 – Leukotriene A4 hydrolase
Handbook of Proteolytic Enzymes, 2004Co-Authors: Jesper Z. HaeggströmAbstract:Publisher Summary This chapter describes structural chemistry and the biological aspects of Leukotriene A4 hydrolase (LTA4). Both catalytic activities of LTA4 hydrolase are dependent on the catalytic zinc atom and are inhibited by divalent cations with different specificity and potency for each of the two activities. In addition, the peptidase activity is greatly stimulated by monovalent anions, including chloride. Typically, LTA4 hydrolase undergoes suicide inactivation when exposed to its lipid substrate LTA4. During this process, both activities are affected to the same degree. The epoxide hydrolase activity of LTA4 hydrolase is very substrate specific and, besides LTA4, the only other known substrates are the double-bond isomers LTA5 and to a lesser extent LTA3. This activity is measured by incubation with the free acid of LTA4, under conditions compatible with its chemical lability, followed by extraction. LTA4 hydrolase from humans and rodents is a soluble monomeric enzyme composed of 610 amino acids with a calculated MΓ of 69 153. The cDNAs encoding human, mouse, rat, guinea pig and Saccharomyces cerevisiae LTA4 hydrolase have been cloned and sequenced.
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Leukotriene A4 hydrolase.
Prostaglandins & Other Lipid Mediators, 2002Co-Authors: Jesper Z. Haeggström, Fredrik Tholander, Filippa Kull, Peter C. Rudberg, Marjolein M. G. M. ThunnissenAbstract:The Leukotrienes (LTs) are a family of lipid mediators involved in inflammation and allergy. Leukotriene B4 is a classical chemoattractant, which triggers adherence and aggregation of leukocytes to the endothelium at only nanomolar concentrations. In addition, Leukotriene B4 modulates immune responses, participates in the host-defense against infections, and is a key mediator of PAF-induced lethal shock. Because of these powerful biological effects, Leukotriene B4 is implicated in a variety of acute and chronic inflammatory diseases, e.g. nephritis, arthritis, dermatitis, and chronic obstructive pulmonary disease. The final step in the biosynthesis of Leukotriene B4 is catalyzed by Leukotriene A4 hydrolase, a unique bi-functional zinc metalloenzyme with an anion-dependent aminopeptidase activity. Here we describe the most recent developments regarding our understanding of the structure, function, and catalytic mechanisms of Leukotriene A4 hydrolase.
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Leukotriene A4 hydrolase.
Prostaglandins & Other Lipid Mediators, 2002Co-Authors: Jesper Z. Haeggström, Fredrik Tholander, Filippa Kull, Peter C. Rudberg, Marjolein M. G. M. ThunnissenAbstract:The Leukotrienes (LT) are bioactive fatty acids defining a subset of a family of molecules collectively known as the eicosanoids, all biologically very active derivatives of carbon-20 fatty acids, in particular arachidonic acid (AA). Leukotriene B4 (LTB4) is one of the most potent chemoattractants known to date and is generated by the enzyme Leukotriene A4 hydrolase (LTA4H). The main physiological role of LTB4 is to recruit leukocytes to the site of inflammation. Highly elevated tissue levels of LTB4 is pathological and suggested to be an element of certain acute and chronic inflammatory diseases and therefore LTA4H defines an interesting drug target for treating such conditions. This chapter discusses properties of LTA4H, the enzyme catalyzing the final and rate-limiting step in LTB4 biosynthesis. Following an overview, where LTA4H is put into a biological and physiological context, a discussion addressing the biochemical and catalytic properties of the enzyme will be given. The crystal structure of LTA4H was recently solved and special emphasis will therefore be placed on structural properties of the enzyme and the new findings emerging from the structural knowledge. In addition, the molecular evolution of LTA4H will be discussed.
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Functional properties and molecular architecture of Leukotriene A4 hydrolase, a pivotal catalyst of chemotactic Leukotriene formation.
The Scientific World JOURNAL, 2002Co-Authors: Jesper Z. Haeggström, Pär Nordlund, Marjolein M. G. M. ThunnissenAbstract:The Leukotrienes are a family of lipid mediators involved in inflammation and allergy. Leukotriene B4 is a classical chemoattractant, which triggers adherence and aggregation of leukocytes to the endothelium at only nM concentrations. In addition, Leukotriene B4 modulates immune responses, participates in the host defense against infections, and is a key mediator of PAF-induced lethal shock. Because of these powerful biological effects, Leukotriene B4 is implicated in a variety of acute and chronic inflammatory diseases, e.g., nephritis, arthritis, dermatitis, and chronic obstructive pulmonary disease. The final step in the biosynthesis of Leukotriene B4 is catalyzed by Leukotriene A4 hydrolase, a unique bifunctional zinc metalloenzyme with an anion-dependent aminopeptidase activity. Here we describe the most recent developments regarding our understanding of the function and molecular architecture of Leukotriene A4 hydrolase.
Anders Wetterholm - One of the best experts on this subject based on the ideXlab platform.
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Structure and catalytic mechanisms of Leukotriene A4 hydrolase.
Prostaglandins & Other Lipid Mediators, 2007Co-Authors: Jesper Z. Haeggström, Fredrik Tholander, Anders WetterholmAbstract:Abstract Leukotriene A4 hydrolase catalyzes the final and committed step in the biosynthesis of Leukotriene B4, a potent chemotactic agent for neutrophils, eosinophils, monocytes, and T-cells that play key roles in the innate immune response. Recent data strongly implicates Leukotriene B4 in the pathogenesis of cardiovascular diseases, in particular arteriosclerosis, myocardial infarction and stroke. Here, we highlight the most salient features of Leukotriene A4 hydrolase with emphasis on its biochemistry and structure biology.
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Leukotriene A4 hydrolase: A key enzyme in chemotactic Leukotriene formation
Novel Inhibitors of Leukotrienes, 1999Co-Authors: Jesper Z. Haeggström, Anders WetterholmAbstract:Leukotrienes are considered as important mediators in a variety of allergic and inflammatory disorders. As discussed elsewhere in this volume, these lipid mediators exert a wide range of biological effects, for example activation of leukocytes and contraction of smooth muscles, particularly in the airways, and microcirculation. In the biosynthesis of Leukotrienes, free arachidonic acid is converted by 5-lipoxygenase into 5(S)-trans-5,6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid, termed Leukotriene A4 (LTA4). This highly unstable epoxide intermediate may subsequently be hydrolyzed into 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4), a reaction catalyzed by LTA4 hydrolase. Alternatively, LTA4 may be conjugated with glutathione, by LTC4 synthase, to produce 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic acid (LTC4). This review will focus on the biochemistry and molecular biology of LTA4 hydrolase, the enzyme catalyzing the final step in the biosynthesis of the potent chemotaxin LTB4, an important mediator of inflammation.
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Leukotriene A4 Hydrolase: Differential Inhibition of the Catalytic Activities by Divalent Cations
Advances in Experimental Medicine and Biology, 1997Co-Authors: Anders Wetterholm, Jesper Z. HaeggströmAbstract:Leukotriene A4 (LTA4) hydrolase (EC 3.3.2.6) catalyzes the hydrolysis of the unstable epoxide intermediate LTA4 into the dihydroxy acid Leukotriene B4 (LTB4) which is a potent chemotactic stimulus for leukocytes. Previous studies have identified LTA4 hydrolase as a zinc metalloenzyme which also exhibits an anion activated peptidase activity (1). The predicted zinc binding ligands, His-295, His-299 and Glu-318 have been verified by site directed mutagenesis combined with zinc analysis of the purified mutated proteins (2). Furthermore, mutagenetic replacements of glutamic acid 296 in LTA4 hydrolase abrogated only the peptidase activity, suggesting a direct catalytic role for this amino acid in the peptidase but not in the epoxide hydrolase reaction (3).
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Amino hydroxamic acids as potent inhibitors of Leukotriene A4 hydrolase
Bioorganic & Medicinal Chemistry, 1995Co-Authors: J. Heather Hogg, Bengt Samuelsson, Anders Wetterholm, Ian R. Ollmann, Jesper Z. Haeggström, Chi-huey WongAbstract:Leukotriene A4 hydrolase is a zinc-containing enzyme which catalyzes the hydrolysis of LTA4 to LTB4, a proinflammatory mediator. The enzyme also exhibits an aminopeptidase activity. Due to its biological importance, it is of considerable interest to develop selective inhibitors of this enzyme. The design and synthesis of a number of potent beta-amino hydroxylamine and amino hydroxamic acid inhibitors are described here. It was found that having a free amine was essential for high activity. Hydroxylamines were found to be about an order of magnitude less potent than their analogous hydroxamic acids. Our investigation of amino hydroxamic acids as inhibitors of Leukotriene A4 hydrolase has led to the development of hydroxamates 16 and 17, which are among the most potent inhibitors found to date. These, compounds were found to be competitive inhibitors with Ki values of 1.6 nM and 3.4 nM respectively, against the peptidase activity. Inhibitor 16 has an IC50 value of < or = 0.15 microM against the epoxide hydrolase activity and is also potent against the production of LTB4 by isolated polymorphonuclear leukocytes (PMNL) activated with ionophore A23187 (IC50 approximately 0.3 microM).
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Zinc and other divalent cations inhibit purified Leukotriene A4 hydrolase and Leukotriene B4 biosynthesis in human polymorphonuclear leukocytes.
Archives of Biochemistry and Biophysics, 1994Co-Authors: Anders Wetterholm, L. Macchia, Jesper Z. HaeggströmAbstract:Leukotriene A4 hydrolase is a bifunctional metalloenzyme that contains 1 mol of zinc per mole of protein. The primary function of the metal is catalytic and zinc is thus necessary for both its peptidase and its epoxide hydrolase activity. However, at concentrations of zinc exceeding a 1:1 molar ratio (metal:enzyme), we found that zinc acted as an inhibitor with IC50 values of 10 microM for the epoxide hydrolase activity, i.e., the conversion of Leukotriene A4 to Leukotriene B4, and 0.1 microM for the peptidase activity. The inhibition of both enzyme activities could be reversed by treating the enzyme with chelating agents such as EDTA or dipicolinic acid. Several divalent cations, other than zinc, were also found to inhibit Leukotriene A4 hydrolase although with different specificity and potency for the two enzyme activities. Thus, CdSO4 and HgCl2 were effective inhibitors (IC50 approximately 10 microM) of the epoxide hydrolase activity, whereas CoCl2 or MnCl2 were not inhibitory even at concentrations of 1 mM. On the other hand, the peptidase activity was inhibited by CdSO4, NiSO4, HgCl2, MnCl2, CoCl2, and PbNO3, listed in decreasing order of potencies (IC50 0.5-10 microM). In addition, zinc in micromolar concentrations inhibited Leukotriene B4 formation in intact human polymorphonuclear leukocytes stimulated by the calcium ionophore A23187 and cell homogenates incubated with arachidonic acid. However, this effect was not related to inhibition of Leukotriene A4 hydrolase but rather to a direct or indirect inhibitory effect on the enzyme 5-lipoxygenase in isolated leukocytes. In these cells, 15-lipoxygenase activity was also inhibited by zinc (IC50 5 microM), whereas Leukotriene C4 synthase activity in human platelets and rat basophilic leukemia cells was significantly affected only at concentrations > or = 1 mM.
Bengt Samuelsson - One of the best experts on this subject based on the ideXlab platform.
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Cloning of Leukotriene A4 hydrolase cDNA.
Methods in Enzymology, 2004Co-Authors: Olof Rådmark, Bengt Samuelsson, Ji Yi Fu, Colin D. Funk, Shigeo Ohno, Hans Jörnvall, Takashi Matsumoto, Hiroshi Kawasaki, Michiko Minami, Yousuke SeyamaAbstract:Publisher Summary The chapter presents a study on cloning of Leukotriene A 4 hydrolase cDNA. Leukotriene A 4 hydrolase (EC 3.3.2.6) is a soluble epoxide hydrolase, which catalyzes the enzymatic hydrolysis of Leukotriene A4 (LTA4) to the chemotactic agent Leukotriene B 4 . After initial purification and characterization of the enzyme, it was determined to be different from other epoxide hydrolases, and further studies were initiated. The cloning of LTA 4 hydrolase was carried out successfully using two different strategies, they are, screening of λgt11 cDNA libraries (which express recombinant protein) with polyclonal antiserum, and screening of a λgtl0 cDNA library with a single isomer oligonucleotide probe (48-mer) whose structure was based on mammalian codon usage frequencies. Prior to this, screening of plasmid (pBR322) cDNA libraries with mixed shorter oligonucleotide probes (16-mers) was unsuccessful. Thus, the use of better quality cDNA synthesizes, according to the RNase H method in λgt10/11 vectors, as well as the use of more efficient probes were beneficial. In the procedure for screening with 48-mer, a 48-mer oligonucleotide was designed from the amino acid sequence of a cyanogen bromide fragment of LTA hydrolase, according to the mammalian codon usage frequencies.
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Leukotriene A4 Hydrolase, Mutation of Tyrosine 378 Allows Conversion of Leukotriene A4 into an Isomer of Leukotriene B4
Journal of Biological Chemistry, 1996Co-Authors: Martin J Mueller, Bengt Samuelsson, Martina Andberg, Haeggström Jesper Z. SamuelssonAbstract:Abstract Leukotriene A4 hydrolase catalyzes the final step in the biosynthesis of the proinflammatory compound Leukotriene B4, a reaction which is accompanied by suicide inactivation of the enzyme by Leukotriene A4. We have recently reported that Tyr-378 is a major structural determinant for suicide inactivation and that mutation of Tyr-378 into Phe or Gln protects Leukotriene A4 hydrolase from this catalytic restriction (Mueller, M. J., Blomster, M., Opperman, U. C. T., Jornvall, H., Samuelsson, B., and Haeggstrom, J. Z. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5931-5935). In the present study, we show that both [Y378F]- and [Y378Q]Leukotriene A4 hydrolase converts Leukotriene A4 not only into Leukotriene B4 but also into a second, previously unknown, product of the enzyme. From biophysical analyses and comparison with a synthetic standard, the structure of this product was determined to 5S,12R-dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid, i.e. Δ6-trans-Δ8-cis-Leukotriene B4. The relative formation of Δ6-trans-Δ8-cis-Leukotriene B4 versus Leukotriene B4 by [Y378F]- and [Y378Q]Leukotriene A4 hydrolase, was 18% and 32%, respectively. For [Y378F]Leukotriene A4 hydrolase, the turnover of Leukotriene A4 into Leukotriene B4 or Δ6-trans-Δ8-cis-Leukotriene B4 was calculated to 2.5 s−1 which is almost three times the kcat value of the wild type enzyme. Taken together, these findings indicate that Tyr-378 is located at the active site where it assists in the formation of the correct double-bond geometry in the product Leukotriene B4.
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Investigation of the inhibition of Leukotriene A4 hydrolase.
Bioorganic & Medicinal Chemistry, 1995Co-Authors: Ian R. Ollmann, Bengt Samuelsson, Benito Munoz, J. Heather Hogg, Jesper Z. Haeggström, Chi-huey WongAbstract:In an effort to better understand the favorable binding interactions between the reversible picomolar inhibitor 3-(4-benzyloxyphenyl)-2-(R)-amino-1-propanethiol (1) and Leukotriene A4 (LTA4) hydrolase (EC 3.3.2.6), we prepared a number of derivatives of 1-l and other related structures, and assayed their inhibition of LTA4 hydrolase-catalyzed hydrolysis of l-alanine-p-nitroanilide. The inhibition data was analyzed using a weighted non-linear least-squares curve fitting computer program developed for this purpose to fit data derived under the non-Michaelis-Menten condition of [I]t < [E]t. The free thiol is necessary for sub-micromolar binding and the enzyme prefers the R enantiomer over the S enantiomer, in contrast to the stereoselectivity displayed towards bestatin, an inhibitor of somewhat similar structure. Substitution of acid moieties around the periphery of the benzyloxyphenyl portion of 1-l leads to substantially decreased binding, suggesting that this group resides within a large hydrophobic pocket when bound to the enzyme. Possible LTA4 binding modes in the active site of LTA4 hydrolase, including a possible direct role for the carboxylic acid of LTA4 in the enzyme-catalyzed hydrolysis of Leukotriene A4, are discussed.
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Chemical modification of Leukotriene A4 hydrolase. Indications for essential tyrosyl and arginyl residues at the active site.
Biochemistry, 1995Co-Authors: Martin J Mueller, Bengt Samuelsson, Jesper Z. HaeggströmAbstract:: We have employed chemical modification to identify amino acids essential for the catalytic activities of the bifunctional zinc metalloenzyme Leukotriene A4 hydrolase (EC 3.3.2.6). The epoxide hydrolase and the peptidase activity were both rapidly inactivated by N-acetylimidazole and tetranitromethane. Furthermore, treatment with 2,3-butanedione and phenylglyoxal also resulted in loss of both activities. Leukotriene A4 hydrolase could be protected from inactivation by these tyrosyl and arginyl reagents by the competitive inhibitors bestatin and captopril, respectively. Two tyrosyl and three arginyl residues were found by differential labeling techniques to be protected by the inhibitors, which thus suggested that these amino acids are located close to or at the active center of the enzyme. Limited modification by thiol reagents and particularly methyl methanethiosulfonate led to a > 10-fold increase in the peptidase activity and a decreased epoxide hydrolase activity, whereas prolonged treatment inhibited both activities. Kinetic analysis of modified enzyme, using the substrate alanine p-nitroanilide, revealed that the stimulatory effect on the peptidase activity was due to increased enzyme displayed a reduced apparent affinity constant for chloride ions, which strongly stimulate the peptidase activity. Neither activation nor inactivation by methyl methanethiosulfonate was influenced by the presence of competitive inhibitors, which suggested that this compound did not react with amino acids at the active center but rather with residues of importance for the overall enzyme conformation.
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Amino hydroxamic acids as potent inhibitors of Leukotriene A4 hydrolase
Bioorganic & Medicinal Chemistry, 1995Co-Authors: J. Heather Hogg, Bengt Samuelsson, Anders Wetterholm, Ian R. Ollmann, Jesper Z. Haeggström, Chi-huey WongAbstract:Leukotriene A4 hydrolase is a zinc-containing enzyme which catalyzes the hydrolysis of LTA4 to LTB4, a proinflammatory mediator. The enzyme also exhibits an aminopeptidase activity. Due to its biological importance, it is of considerable interest to develop selective inhibitors of this enzyme. The design and synthesis of a number of potent beta-amino hydroxylamine and amino hydroxamic acid inhibitors are described here. It was found that having a free amine was essential for high activity. Hydroxylamines were found to be about an order of magnitude less potent than their analogous hydroxamic acids. Our investigation of amino hydroxamic acids as inhibitors of Leukotriene A4 hydrolase has led to the development of hydroxamates 16 and 17, which are among the most potent inhibitors found to date. These, compounds were found to be competitive inhibitors with Ki values of 1.6 nM and 3.4 nM respectively, against the peptidase activity. Inhibitor 16 has an IC50 value of < or = 0.15 microM against the epoxide hydrolase activity and is also potent against the production of LTB4 by isolated polymorphonuclear leukocytes (PMNL) activated with ionophore A23187 (IC50 approximately 0.3 microM).
Martina Andberg - One of the best experts on this subject based on the ideXlab platform.
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Analysis of the Molecular Mechanism of Substrate-mediated Inactivation of Leukotriene A4 Hydrolase
Journal of Biological Chemistry, 1998Co-Authors: Martin J Mueller, Martina Andberg, Jesper Z. HaeggströmAbstract:Abstract The bifunctional Leukotriene A4hydrolase catalyzes the final step in the biosynthesis of the proinflammatory Leukotriene B4. During exposure to the substrate Leukotriene A4, a labile allylic epoxide, the enzyme is gradually inactivated as a consequence of the covalent binding of Leukotriene A4 to the active site. This phenomenon, commonly referred to as suicide inactivation, has previously been rationalized as a mechanism-based process in which the enzyme converts the substrate to a highly reactive intermediate within an activated enzyme-substrate complex that partitions between covalent bond formation (inactivation) and catalysis. To further explore the molecular mechanism of the self-inactivation of Leukotriene A4 hydrolase by Leukotriene A4, we prepared and analyzed mutated forms of the enzyme that were either catalytically incompetent or fully active but resistant toward substrate-mediated inactivation. These mutants were treated with Leukotriene A4 and Leukotriene A4 methyl and ethyl esters and subjected to differential peptide mapping and enzyme activity determinations, which showed that inactivation and/or covalent modification can be completely dissociated from catalysis. Our results, together with recent findings described in the literature, argue against a mechanism-based model for suicide inactivation. We conclude that the collected data on the substrate-mediated inactivation of Leukotriene A4 hydrolase best conforms to an affinity-labeling mechanism.
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Mutation of tyrosine 383 in Leukotriene A4 hydrolase allows conversion of Leukotriene A4 into 5S,6S-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid. Implications for the epoxide hydrolase mechanism.
Journal of Biological Chemistry, 1997Co-Authors: Martina Andberg, Mats Hamberg, Jesper Z. HaeggströmAbstract:Abstract Leukotriene A4 hydrolase is a bifunctional zinc metalloenzyme that catalyzes the final step in the biosynthesis of the proinflammatory mediator Leukotriene B4. In previous studies with site-directed mutagenesis on mouse Leukotriene A4 hydrolase, we have identified Tyr-383 as a catalytic amino acid involved in the peptidase reaction. Further characterization of the mutants in position 383 revealed that [Y383H], [Y383F], and [Y383Q] Leukotriene A4hydrolases catalyzed hydrolysis of Leukotriene A4 into a novel enzymatic metabolite. From analysis by high performance liquid chromatography, gas chromatography/mass spectrometry of material generated in the presence of H2 16O or H2 18O, steric analysis of the hydroxyl groups, treatment with soybean lipoxygenase, and comparison with a synthetic standard, the novel metabolite was assigned the structure 5S,6S-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid (5S,6S-DHETE). The kinetic parameters for the formation of 5S,6S-DHETE and Leukotriene B4 were found to be similar. Also, both activities were susceptible to suicide inactivation and were equally sensitive to inhibition by bestatin. Moreover, from the stereochemical configuration of the vicinal diol, it could be inferred that 5S,6S-DHETE is formed via an SN1 mechanism involving a carbocation intermediate, which in turn indicates that enzymatic hydrolysis of Leukotriene A4 into Leukotriene B4 follows the same mechanism. Inasmuch as soluble epoxide hydrolase utilizes Leukotriene A4 as substrate to produce 5S,6R-DHETE, our results also suggest a functional relationship between Leukotriene A4 hydrolase and xenobiotic epoxide hydrolases.
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Mutation of Tyrosine 383 in Leukotriene A4Hydrolase Allows Conversion of Leukotriene A4 into 5S,6S-Dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic Acid IMPLICATIONS FOR THE EPOXIDE HYDROLASE MECHANISM
Journal of Biological Chemistry, 1997Co-Authors: Martina Andberg, Mats Hamberg, Jesper Z. HaeggströmAbstract:Abstract Leukotriene A4 hydrolase is a bifunctional zinc metalloenzyme that catalyzes the final step in the biosynthesis of the proinflammatory mediator Leukotriene B4. In previous studies with site-directed mutagenesis on mouse Leukotriene A4 hydrolase, we have identified Tyr-383 as a catalytic amino acid involved in the peptidase reaction. Further characterization of the mutants in position 383 revealed that [Y383H], [Y383F], and [Y383Q] Leukotriene A4hydrolases catalyzed hydrolysis of Leukotriene A4 into a novel enzymatic metabolite. From analysis by high performance liquid chromatography, gas chromatography/mass spectrometry of material generated in the presence of H2 16O or H2 18O, steric analysis of the hydroxyl groups, treatment with soybean lipoxygenase, and comparison with a synthetic standard, the novel metabolite was assigned the structure 5S,6S-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid (5S,6S-DHETE). The kinetic parameters for the formation of 5S,6S-DHETE and Leukotriene B4 were found to be similar. Also, both activities were susceptible to suicide inactivation and were equally sensitive to inhibition by bestatin. Moreover, from the stereochemical configuration of the vicinal diol, it could be inferred that 5S,6S-DHETE is formed via an SN1 mechanism involving a carbocation intermediate, which in turn indicates that enzymatic hydrolysis of Leukotriene A4 into Leukotriene B4 follows the same mechanism. Inasmuch as soluble epoxide hydrolase utilizes Leukotriene A4 as substrate to produce 5S,6R-DHETE, our results also suggest a functional relationship between Leukotriene A4 hydrolase and xenobiotic epoxide hydrolases.
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Leukotriene A4 Hydrolase, Mutation of Tyrosine 378 Allows Conversion of Leukotriene A4 into an Isomer of Leukotriene B4
Journal of Biological Chemistry, 1996Co-Authors: Martin J Mueller, Bengt Samuelsson, Martina Andberg, Haeggström Jesper Z. SamuelssonAbstract:Abstract Leukotriene A4 hydrolase catalyzes the final step in the biosynthesis of the proinflammatory compound Leukotriene B4, a reaction which is accompanied by suicide inactivation of the enzyme by Leukotriene A4. We have recently reported that Tyr-378 is a major structural determinant for suicide inactivation and that mutation of Tyr-378 into Phe or Gln protects Leukotriene A4 hydrolase from this catalytic restriction (Mueller, M. J., Blomster, M., Opperman, U. C. T., Jornvall, H., Samuelsson, B., and Haeggstrom, J. Z. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5931-5935). In the present study, we show that both [Y378F]- and [Y378Q]Leukotriene A4 hydrolase converts Leukotriene A4 not only into Leukotriene B4 but also into a second, previously unknown, product of the enzyme. From biophysical analyses and comparison with a synthetic standard, the structure of this product was determined to 5S,12R-dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid, i.e. Δ6-trans-Δ8-cis-Leukotriene B4. The relative formation of Δ6-trans-Δ8-cis-Leukotriene B4 versus Leukotriene B4 by [Y378F]- and [Y378Q]Leukotriene A4 hydrolase, was 18% and 32%, respectively. For [Y378F]Leukotriene A4 hydrolase, the turnover of Leukotriene A4 into Leukotriene B4 or Δ6-trans-Δ8-cis-Leukotriene B4 was calculated to 2.5 s−1 which is almost three times the kcat value of the wild type enzyme. Taken together, these findings indicate that Tyr-378 is located at the active site where it assists in the formation of the correct double-bond geometry in the product Leukotriene B4.
Giancarlo Folco - One of the best experts on this subject based on the ideXlab platform.
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Transcellular metabolism of Leukotriene A4 by rabbit blood cells: lack of relevant LTC4-synthase activity in rabbit platelets.
Journal of lipid research, 1997Co-Authors: Angelo Sala, T. Testa, F Nobili, Giancarlo FolcoAbstract:The objective of this study was to determine the transcellular metabolism of Leukotriene A4 by rabbit blood cells. mixed peripheral blood leukocyte preparations with and without platelets in a ratio of 1:40 were challenged with the Ca(2+)-ionophore A23187. 5-Lipoxygenase metabolites production was assessed by RP-HPLC coupled with diode-array UV detection. In light of the observation that Leukotriene C4 production in leukocyte-platelet coincubation was the same as with leukocytes alone, mixed coincubation of human and rabbit blood cells was tested. Rabbit leukocytes with human platelets resulted in a significant increase of Leukotriene C4 production, while no changes were observed in human leukocytes with or without rabbit platelets. In agreement with these results, intact rabbit platelets or rabbit platelet lysates, unlike human platelets, were not able to convert synthetic Leukotriene A4 free acid to Leukotriene C4. These data provide evidence that rabbit leukocytes are able to make a significant amount of Leukotriene A4 available for transcellular metabolism, while rabbit platelets, unlike human platelets, lack Leukotriene C4-synthase activity.
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Release of Leukotriene A4 Versus Leukotriene B4 from Human Polymorphonuclear Leukocytes
Journal of Biological Chemistry, 1996Co-Authors: Angelo Sala, Manlio Bolla, Simona Zarini, Reiner Muller-peddinghaus, Giancarlo FolcoAbstract:Abstract The reactive intermediate formed by 5-lipoxygenase metabolism of arachidonic acid, Leukotriene A4, is known to be released from cells and subsequently taken up by other cells for biochemical processing. The objective of this study was to determine the relative amount of Leukotriene A4 synthesized by human polymorphonuclear leukocytes (PMNL) that is available for transcellular biosynthetic processes. This was accomplished by diluting cell suspensions and measuring the relative amounts of enzymatic versus nonenzymatic Leukotriene A4-derived metabolites after challenge with the Ca2+ ionophore A23187. Nonenzymatic Leukotriene A4-derived metabolites were used as a quantitative index of the amount of Leukotriene A4 released into the extracellular milieu. The results obtained demonstrated that in human PMNL, the relative amounts of nonenzymatic versus enzymatic Leukotriene A4-derived metabolites increased with decreasing cell concentrations. After a 20-fold dilution of PMNL in cell preparations, a doubling in the amount of nonenzymatic Leukotriene A4-derived metabolites was observed following challenge (from 53.9 ± 1.3 to 110.4 ± 8.9 pmol/106 PMNL, p < 0.01). Reduction of possible cell-cell interactions by dilution suggested that over 50% of Leukotriene A4 synthesized is released from the PMNL. These data provide evidence that, in human PMNL preparations, transfer of Leukotriene A4 to neighboring PMNL is taking place, resulting in additional formation of Leukotriene B4 and its ω-oxidized metabolites 20-hydroxy- and 20-carboxy-Leukotriene B4. Neutrophil reuptake of extracellular Leukotriene A4 leads to an underestimation of the fraction of Leukotriene A4 that is in fact available for transcellular metabolism when tight cell-cell interactions occur, such as during PMNL adhesion to the microvascular endothelium and diapedesis.