This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A bromoenol lactone suicide substrate inactivates Group VIA phospholipase A2 by generating a diffusible bromomethyl ketoacid that alkylates cysteine thiols: Phospholipases A2 (PLA2) comprise a superfamily of enzymes that hydrolyze phospholipids to a free fatty acid, e.g., arachidonate, and a 2-lysophospholipid. Dissecting their individual functions has relied in large part on pharmacological inhibitors that discriminate among PLA2. Group VIA PLA2 (iPLA2 ) has a GTSTG serine lipase consensus sequence, and studies with a bromoenol lactone (BEL) suicide substrate inhibitor suggest that iPLA2 participates in a wide variety of biological processes. Inhibition by BEL requires its hydrolysis by and results in uncharacterized covalent modification(s) of iPLA2 . We performed mass spectrometric analyses of proteolytic digests of BEL-treated iPLA2 to identify modifications associated with loss of activity. The GTSTG active site and large flanking regions of sequence are not modified by BEL-treatment, but most iPLA2 Cys residues are alkylated at various [BEL] to form a thioether linkage to a BEL ketoacid hydrolysis product. Synthetic Cys-containing peptides are alkylated when incubated with iPLA2 and BEL, which reflects iPLA2 -catalyzed BEL hydrolysis to a diffusible bromomethyl-ketoacid product that reacts with distant thiols. Cys651 alkylation is strongly associated with loss of iPLA2b activity, and no amino acid residues other than Cys were found to be modified, suggesting that Cys651 might play a previously unsuspected role in catalysis, substrate recognition, or maintaining an active conformation of iPLA2 .
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