LEUKOTRIENE A(4) HYDROLASE - MAPPING OF A HENICOSAPEPTIDE INVOLVED INMECHANISM-BASED INACTIVATION

Leukotriene A(4) (LTA(4)) hydrolase [(7E,9E,11Z, 14Z)-(5S,6S)-5,6-epox yicosa-7,9,11,14-tetraenoate hydrolase; EC 3.3.2.6] is a bifunctional zinc metalloenzyme which converts LTA(4) into the chemotactic agent le ukotriene B-4 (LTB(4)). Suicide inactivation, a typical feature of LTA (4) hydrolase/aminopeptidase, occurs via an irreversible, apparently m echanism-based, covalent binding of LTA(4) to the protein in a 1:1 sto ichiometry. Differential lysine-specific peptide mapping of unmodified and suicide-inactivated LTA(4) hydrolase has been used to identify a henicosapeptide, encompassing the amino acid residues 365-385 of human LTA(4) hydrolase, which is involved in the binding of LTA4, LTA(4) me thyl ester, and LTA(4) ethyl ester to the native enzyme, A modified fo rm of this peptide, generated by lysine-specific digestion of LTA(4) h ydrolase inactivated by LTA(4) ethyl ester, could be isolated for comp lete Edman degradation, The sequence analysis revealed a gap at positi on 14, which shows that binding of the leukotriene epoxide had occurre d via Tyr-378 in LTA(4) hydrolase, Inactivation of the epoxide hydrola se acid the aminopeptidase activity was accompanied by a proportionate modification of the peptide. Furthermore, both enzyme inactivation an d peptide modification could be prevented by preincubation of LTA(4) h ydrolase with the competitive inhibitor bestatin, which demonstrates t hat the henicosapeptide contains functional elements of the active sit e(s), It may now be possible to clarify the molecular mechanisms under lying suicide inactivation and epoxide hydrolysis by site-directed mut agenesis combined with structural analysis of the lipid molecule, cova lently bound to the peptide.