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

M.B. Andberg, M. Hamberg, J.Z. Haeggström

Research output: Contribution to journalArticleScientificpeer-review

24 Citations (Scopus)

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.
Original languageEnglish
Pages (from-to)23057-23063
JournalJournal of Biological Chemistry
Volume272
DOIs
Publication statusPublished - 1997
MoE publication typeA1 Journal article-refereed

Fingerprint

NSC 153174
Leukotriene A4
Arachidonic Acids
Epoxide Hydrolases
Leukotriene B4
Tyrosine
Metabolites
Mutation
Hydrolysis
Mutagenesis
Enzymatic hydrolysis
Lipoxygenase
Leukotrienes
Biosynthesis
High performance liquid chromatography
Xenobiotics
Site-Directed Mutagenesis
Soybeans
Kinetic parameters
Gas chromatography

Cite this

@article{ca1dd8b03bb74723a5adc83f9660b048,
title = "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",
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.",
author = "M.B. Andberg and M. Hamberg and J.Z. Haeggstr{\"o}m",
year = "1997",
doi = "10.1074/jbc.272.37.23057",
language = "English",
volume = "272",
pages = "23057--23063",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",

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T1 - 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

T2 - Implications for the epoxide hydrolase mechanism

AU - Andberg, M.B.

AU - Hamberg, M.

AU - Haeggström, J.Z.

PY - 1997

Y1 - 1997

N2 - 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.

AB - 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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U2 - 10.1074/jbc.272.37.23057

DO - 10.1074/jbc.272.37.23057

M3 - Article

VL - 272

SP - 23057

EP - 23063

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

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