Structural Characterization of the Covalent Attachment of Leukotriene A3to Leukotriene A4Hydrolase

Leukotriene A4(LTA4) hydrolase catalyzes the conversion of the unstable epoxide LTA4[5(S)-trans-5,6-oxido-11,14-cis-eicosatetraenoic acid] into proinflammatory LTB4. During the process of catalyzing this reaction, the enzyme is suicide inactivated by its substrate. In addition, LTA3, an analogue of LTA4that lacks the C14–C15double bond, is a potent suicide inhibitor of LTA4hydrolase. We have synthesized [3H]LTA3and used this ligand to demonstrate that LTA3can covalently label LTA4hydrolase and that this labeling is specifically competed for by bestatin and LTA4. Incubation of recombinant human LTA4hydrolase with LTA3followed by proteolysis (endoproteinase Lys-C) resulted in a peptide map with a single modified peptide defining the location of the LTA3covalent attachment region. This modified 21-amino-acid peptide had a UV absorption spectrum corresponding to a conjugated triene chromophore which established conservation of this structural unit after covalent interaction of LTA3with LTA4hydrolase. MALDI-TOF mass spectrometric analysis of the 21-amino-acid peptide adduct revealed an abundant MH+atm/z2658, consistent with the predicted nominal mass of the sequenced peptide with the addition of a single LTA3moiety. Proteolysis of LTA4hydrolase modified with LTA3was performed sequentially with endo-Asp-N and endo-Lys-C. The resulting peptide isolated by reverse-phase high-performance liquid chromatography was analyzed by mass spectroscopy revealing two related peptides, D371-K385 (m/z2018.0) and D375-K385 (m/z1577.8), both of which retained the elements of LTA3. Postsource decay ofm/z1577.8 resulted in an abundant ion atm/z536 and an ion of lesser abundance atm/z856 consistent with cleavage between V381 and P382 that supported assignment of the modified tyrosine residue at Y383. These results suggest nucleophilic attack of a tyrosine residue (Y383) at the conjugated triene epoxide of LTA3resulting in a triene ether carbinol covalent adduct.