MUTATION OF TYROSINE-383 IN LEUKOTRIENE A(4) HYDROLASE ALLOWS CONVERSION OF LEUKOTRIENE A(4) INTO ,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 c atalyzes the final step in the biosynthesis of the proinflammatory med iator leukotriene B-4. In previous studies with site-directed mutagene sis on mouse leukotriene A(4) hydrolase, we have identified Tyr-383 as a catalytic amino acid involved in the peptidase reaction. Further ch aracterization of the mutants in position 383 revealed that [Y383H], [ Y383F], and [Y383Q] leukotriene A(4) hydrolases catalyzed hydrolysis o f leukotriene A(4) into a novel enzymatic metabolite. From analysis by high performance liquid chromatography, gas chromatography/mass spect rometry of material generated in the presence of (H2O)-O-16 or (H2O)-O -18, steric analysis of the hydroxyl groups, treatment with soybean li poxygenase, and comparison with a synthetic standard, the novel metabo lite was assigned the structure 5S,6S-dihydroxy-7,9-trans- 11,14-cis-e icosatetraenoic acid (5S,6S-DHETE). The kinetic parameters for the for mation of 5S,6S-DHETE and leukotriene B-4 were found to be similar. Al so, both activities were susceptible to suicide inactivation and were equally sensitive to inhibition by bestatin. Moreover, from the stereo chemical configuration of the vicinal diol, it could be inferred that 5S,6S-DHETE is formed via an S(N)1 mechanism involving a carbocation i ntermediate, which in turn indicates that enzymatic hydrolysis of leuk otriene A(4) into leukotriene B-4 follows the same mechanism. Inasmuch as soluble epoxide hydrolase utilizes leukotriene A(4) as substrate t o produce 5S,6R-DHETE, our results also suggest a functional relations hip between leukotriene A(4) hydrolase and xenobiotic epoxide hydrolas es.