Leukotriene A4 hydrolase: protection from mechanism-based inactivation by mutation of tyrosine-378.
Leukotriene A4 (LTA4) hydrolase [(7E,9E,11Z,14Z)-(5S,6S)-5,6-epoxyicosa-7, 9,11,14-tetraenoate hydrolase; EC 3.3.2.6] is a bifunctional zinc metalloenzyme that catalyzes the final step in the biosynthesis of the potent chemotactic agent leukotriene B4 (LTB4). LTA4 hydrolase/aminopeptidase is suicide...
| Main Authors: | , , , , , |
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| Formato: | Journal article |
| Idioma: | English |
| Publicado em: |
1996
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| _version_ | 1826282378393288704 |
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| author | Mueller, M Blomster, M Oppermann, U Jörnvall, H Samuelsson, B Haeggström, J |
| author_facet | Mueller, M Blomster, M Oppermann, U Jörnvall, H Samuelsson, B Haeggström, J |
| author_sort | Mueller, M |
| collection | OXFORD |
| description | Leukotriene A4 (LTA4) hydrolase [(7E,9E,11Z,14Z)-(5S,6S)-5,6-epoxyicosa-7, 9,11,14-tetraenoate hydrolase; EC 3.3.2.6] is a bifunctional zinc metalloenzyme that catalyzes the final step in the biosynthesis of the potent chemotactic agent leukotriene B4 (LTB4). LTA4 hydrolase/aminopeptidase is suicide inactivated during catalysis via an apparently mechanism-based irreversible binding of LTA4 to the protein in a 1:1 stoichiometry. Previously, we have identified a henicosapeptide, encompassing residues Leu-365 to Lys-385 in human LTA4 hydrolase, which contains a site involved in the covalent binding of LTA4 to the native enzyme. To investigate the role of Tyr-378, a potential candidate for this binding site, we exchanged Tyr for Phe or Gln in two separate mutants. In addition, each of two adjacent and potentially reactive residues, Ser-379 and Ser-380, were exchanged for Ala. The mutated enzymes were expressed as (His)6-tagged fusion proteins in Escherichia coli, purified to apparent homogeneity, and characterized. Enzyme activity determinations and differential peptide mapping, before and after repeated exposure to LTA4, revealed that wild-type enzyme and the mutants [S379A] and [S380A]LTA4hydrolase were equally susceptible to suicide inactivation whereas the mutants in position 378 were no longer inactivated or covalently modified by LTA4. Furthermore, in [Y378F]LTA4 hydrolase, the value of kcat for epoxide hydrolysis was increased 2.5-fold over that of the wild-type enzyme. Thus, by a single-point mutation in LTA4 hydrolase, catalysis and covalent modification/inactivation have been dissociated, yielding an enzyme with increased turnover and resistance to mechanism-based inactivation. |
| first_indexed | 2024-03-07T00:42:56Z |
| format | Journal article |
| id | oxford-uuid:83ac3ba4-1d38-4a1e-8623-9e6f54ff15f4 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T00:42:56Z |
| publishDate | 1996 |
| record_format | dspace |
| spelling | oxford-uuid:83ac3ba4-1d38-4a1e-8623-9e6f54ff15f42022-03-26T21:45:49ZLeukotriene A4 hydrolase: protection from mechanism-based inactivation by mutation of tyrosine-378.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:83ac3ba4-1d38-4a1e-8623-9e6f54ff15f4EnglishSymplectic Elements at Oxford1996Mueller, MBlomster, MOppermann, UJörnvall, HSamuelsson, BHaeggström, JLeukotriene A4 (LTA4) hydrolase [(7E,9E,11Z,14Z)-(5S,6S)-5,6-epoxyicosa-7, 9,11,14-tetraenoate hydrolase; EC 3.3.2.6] is a bifunctional zinc metalloenzyme that catalyzes the final step in the biosynthesis of the potent chemotactic agent leukotriene B4 (LTB4). LTA4 hydrolase/aminopeptidase is suicide inactivated during catalysis via an apparently mechanism-based irreversible binding of LTA4 to the protein in a 1:1 stoichiometry. Previously, we have identified a henicosapeptide, encompassing residues Leu-365 to Lys-385 in human LTA4 hydrolase, which contains a site involved in the covalent binding of LTA4 to the native enzyme. To investigate the role of Tyr-378, a potential candidate for this binding site, we exchanged Tyr for Phe or Gln in two separate mutants. In addition, each of two adjacent and potentially reactive residues, Ser-379 and Ser-380, were exchanged for Ala. The mutated enzymes were expressed as (His)6-tagged fusion proteins in Escherichia coli, purified to apparent homogeneity, and characterized. Enzyme activity determinations and differential peptide mapping, before and after repeated exposure to LTA4, revealed that wild-type enzyme and the mutants [S379A] and [S380A]LTA4hydrolase were equally susceptible to suicide inactivation whereas the mutants in position 378 were no longer inactivated or covalently modified by LTA4. Furthermore, in [Y378F]LTA4 hydrolase, the value of kcat for epoxide hydrolysis was increased 2.5-fold over that of the wild-type enzyme. Thus, by a single-point mutation in LTA4 hydrolase, catalysis and covalent modification/inactivation have been dissociated, yielding an enzyme with increased turnover and resistance to mechanism-based inactivation. |
| spellingShingle | Mueller, M Blomster, M Oppermann, U Jörnvall, H Samuelsson, B Haeggström, J Leukotriene A4 hydrolase: protection from mechanism-based inactivation by mutation of tyrosine-378. |
| title | Leukotriene A4 hydrolase: protection from mechanism-based inactivation by mutation of tyrosine-378. |
| title_full | Leukotriene A4 hydrolase: protection from mechanism-based inactivation by mutation of tyrosine-378. |
| title_fullStr | Leukotriene A4 hydrolase: protection from mechanism-based inactivation by mutation of tyrosine-378. |
| title_full_unstemmed | Leukotriene A4 hydrolase: protection from mechanism-based inactivation by mutation of tyrosine-378. |
| title_short | Leukotriene A4 hydrolase: protection from mechanism-based inactivation by mutation of tyrosine-378. |
| title_sort | leukotriene a4 hydrolase protection from mechanism based inactivation by mutation of tyrosine 378 |
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