| 1. |
- Haeggstrom, JZ, et al.
(författare)
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Leukotriene A4 hydrolase
- 2002
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Ingår i: Prostaglandins & other lipid mediators. - : Elsevier BV. - 1098-8823. ; 68-968-69, s. 495-510
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Tidskriftsartikel (refereegranskat)
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| 2. |
- Haeggström, JZ, et al.
(författare)
-
Leukotriene A4 hydrolase
- 2002
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Ingår i: Prostaglandins & other lipid mediators. - : Elsevier BV. - 1098-8823. ; 68-69, s. 495-510
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Tidskriftsartikel (refereegranskat)
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| 3. |
- Tholander, F, et al.
(författare)
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Leukotriene A4 Hydrolase, Insights into the Molecular Evolution by Homology Modeling and Mutational Analysis of Enzyme from Saccharomyces cerevisiae
- 2005
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Ingår i: Journal of Biological Chemistry. - 1083-351X .- 0021-9258. ; 280:39, s. 33477-33486
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Rudberg, P C, et al.
(författare)
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Leukotriene A(4) hydrolase - Identification of a common carboxylate recognition site for the epoxide hydrolase and aminopeptidase substrates
- 2004
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Ingår i: Journal of Biological Chemistry. - 1083-351X .- 0021-9258. ; 279:26, s. 27376-27382
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Tidskriftsartikel (refereegranskat)abstract
- Leukotriene ( LT) A(4) hydrolase is a bifunctional zinc metalloenzyme, which converts LTA(4) into the neutrophil chemoattractant LTB4 and also exhibits an anion-dependent aminopeptidase activity. In the x-ray crystal structure of LTA(4) hydrolase, Arg(563) and Lys(565) are found at the entrance of the active center. Here we report that replacement of Arg(563), but not Lys(565), leads to complete abrogation of the epoxide hydrolase activity. However, mutations of Arg(563) do not seem to affect substrate binding strength, because values of K-i for LTA(4) are almost identical for wild type and ( R563K) LTA(4) hydrolase. These results are supported by the 2.3-Angstrom crystal structure of (R563A) LTA(4) hydrolase, which does not reveal structural changes that can explain the complete loss of enzyme function. For the aminopeptidase reaction, mutations of Arg(563) reduce the catalytic activity (V-max = 0.3 - 20%), whereas mutations of Lys(565) have limited effect on catalysis (V-max = 58 - 108%). However, in (K565A)- and (K565M) LTA(4) hydrolase, i.e. mutants lacking a positive charge, values of the Michaelis constant for alanine-p-nitroanilide increase significantly (K-m = 480 - 640%). Together, our data indicate that Arg(563) plays an unexpected, critical role in the epoxide hydrolase reaction, presumably in the positioning of the carboxylate tail to ensure perfect substrate alignment along the catalytic elements of the active site. In the aminopeptidase reaction, Arg(563) and Lys(565) seem to cooperate to provide sufficient binding strength and productive alignment of the substrate. In conclusion, Arg(563) and Lys(565) possess distinct roles as carboxylate recognition sites for two chemically different substrates, each of which is turned over in separate enzymatic reactions catalyzed by LTA(4) hydrolase.
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