Structural characterization of the covalent attachment of leukotriene A3 to leukotriene A4 hydrolase

Leukotriene A(4) (LTA(4)) hydrolase catalyzes the conversion of the unstable epoxide LTA(4) [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, LTA(3), an analogue of LTA(4) that lacks the C-14-C-15 double bond, is a potent suicide inhibitor of LTA(4) hydrolase, We have synthesized [H-3]LTA(3) and used this ligand to demonstrate that LTA(3) can covalently label LTA(4) hydrolase and that this labeling is specifically competed for by bestatin and LTA(3), Incubation of recombinant human LTA(4) hydrolase with LTA(3) followed by proteolysis (endoproteinase Lys-C) resulted in a peptide map with a single modified peptide defining the location of the LTA(3) covalent attachment region, This modified al-aminoacid peptide had a UV absorption spectrum corresponding to a conjugated triene chromophore which established conservation of this structural unit after covalent interaction of LTA(3) with LTA(4) hydrolase, MALDI-TOF mass spectrometric analysis of the 21-amino-acid peptide adduct revealed an abundant MH+ at mit 2658, consistent with the predicted nominal mass of the sequenced peptide with the addition of a single LTA(3) moiety, Proteolysis of LTA(4) hydrolase modified with LTA(3) was 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/z 2018.0) and D375-K385 (m/z 1577.8), both of which retained the elements of LTA(3). Postsource decay of mit 1577.8 resulted in an abundant ion at mit 536 and an ion of lesser abundance at mit 856 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 LTA(3) resulting in a triene ether carbinol covalent adduct. (C) 1998 Academic Press.