STRUCTURAL CHARACTERIZATION OF THE COVALENT ATTACHMENT OF LEUKOTRIENEA(3) TO LEUKOTRIENE A(4) HYDROLASE

Leukotriene A(4) (LTA(4)) hydrolase catalyzes the conversion of the un stable epoxide LTA(4) [5(S)-trans-5,6-oxido-11,14-cis-eicosatetraenoic acid] into proinflammatory LTB4. During the process of catalyzing thi s reaction, the enzyme is suicide inactivated by its substrate. In add ition, LTA(3), an analogue of LTA(4) that lacks the C-14-C-15 double b ond, is a potent suicide inhibitor of LTA(4) hydrolase, We have synthe sized [H-3]LTA(3) and used this ligand to demonstrate that LTA(3) can covalently label LTA(4) hydrolase and that this labeling is specifical ly competed for by bestatin and LTA(3), Incubation of recombinant huma n LTA(4) hydrolase with LTA(3) followed by proteolysis (endoproteinase Lys-C) resulted in a peptide map with a single modified peptide defin ing the location of the LTA(3) covalent attachment region, This modifi ed 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) hydr olase, MALDI-TOF mass spectrometric analysis of the 21-amino-acid pept ide adduct revealed an abundant MH+ at mit 2658, consistent with the p redicted nominal mass of the sequenced peptide with the addition of a single LTA(3) moiety, Proteolysis of LTA(4) hydrolase modified with LT A(3) was performed sequentially with endo-Asp-N and endo-Lys-C, The re sulting peptide isolated by reverse-phase high-performance liquid chro matography was analyzed by mass spectroscopy revealing two related pep tides, D371-K385 (m/z 2018.0) and D375-K385 (m/z 1577.8), both of whic h retained the elements of LTA(3). Postsource decay of mit 1577.8 resu lted in an abundant ion at mit 536 and an ion of lesser abundance at m it 856 consistent with cleavage between V381 and P382 that supported a ssignment of the modified tyrosine residue at Y383, These results sugg est nucleophilic attack of a tyrosine residue (Y383) at the conjugated triene epoxide of LTA(3) resulting in a triene ether carbinol covalen t adduct. (C) 1998 Academic Press.