Mutation of tyrosine 383 in leukotriene A(4) hydrolase allows conversion of leukotriene A(4) into 5S,6S-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid - Implications for the epoxide hydrolase mechanism

Leukotriene A(4) hydrolase is a bifunctional zinc metalloenzyme that catalyzes the final step in the biosynthesis of the proinflammatory mediator leukotriene B-4. In previous studies with site-directed mutagenesis on mouse leukotriene A(4) 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 A(4) hydrolases catalyzed hydrolysis of leukotriene A(4) into a novel enzymatic metabolite. From analysis by high performance liquid chromatography, gas chromatography/mass spectrometry of material generated in the presence of (H2O)-O-16 or (H2O)-O-18, 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 B-4 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 S(N)1 mechanism involving a carbocation intermediate, which in turn indicates that enzymatic hydrolysis of leukotriene A(4) into leukotriene B-4 follows the same mechanism. Inasmuch as soluble epoxide hydrolase utilizes leukotriene A(4) as substrate to produce 5S,6R-DHETE, our results also suggest a functional relationship between leukotriene A(4) hydrolase and xenobiotic epoxide hydrolases.