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 A4 hydrolases 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 H216O or H218O, 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.
Highlights
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 Tyr383 as a catalytic amino acid involved in the peptidase reaction
From the stereochemical configuration of the vicinal diol, it could be inferred that 5S,6SDHETE 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
Summary
From data obtained by site-directed mutagenesis and biochemical analysis of purified recombinant proteins, Glu-296, a residue conserved within the zinc-binding motif, was shown to be catalytic in the peptidase reaction, where it presumably acts as a general base [10]. Such a reaction mechanism postulates a proton transfer to the nitrogen of the peptide bond [11, 12]. Inasmuch as soluble epoxide hydrolase accepts LTA4 as substrate and converts it into an epimeric 5,6-DHETE, our data suggest a functional link between LTA4 hydrolase and xenobiotic soluble epoxide hydrolase
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