AMINO-ACID-RESIDUES OF 5-LIPOXYGENASE-ACTIVATING PROTEIN CRITICAL FORTHE BINDING OF LEUKOTRIENE BIOSYNTHESIS INHIBITORS

5-Lipoxygenase-activating protein (FLAP) plays an essential role in ce llular leukotriene (LT) synthesis and represents the target of three c lasses of LT biosynthesis inhibitors. We have taken three approaches t o localize regions of FLAP involved in the binding of these inhibitors . A comparison of the amino acid sequences of FLAP from eight mammalia n species identifies regions of the protein which are highly conserved and consequently may be involved in functional and inhibitor binding properties of the protein. Conversely, amino acids not conserved among st these species are unlikely to play an essential role in inhibitor b inding. Immunoprecipitation of peptide fragments of FLAP cross-linked to photoaffinity analogues of LT biosynthesis inhibitors following sit e-specific peptide cleavage indicates that the inhibitor attachment si te is amino-terminal to 72Trp. Taken together, the cross-species analy sis and photoaffinity labelling studies suggest a region within the fi rst hydrophilic loop of FLAP which may be important for inhibitor bind ing. Site-directed mutagenesis of human FLAP followed by the analysis of FLAP mutants in a radioligand binding assay was used to more accura tely define critical amino acid residues within this region. Mutagenes is studies reveal that mutants containing deletions of amino acids in regions of FLAP not conserved between species retain the ability to sp ecifically bind inhibitors. furthermore, mutants containing deletions in a highly conserved region of the protein (residues 42-61) do not bi nd inhibitors. These studies have therefore localized specific amino a cids of FLAP which are essential for inhibitor binding. The roles that these amino acids play in inhibitor binding and may play in 5-LO acti vation is under investigation.