A bromoenol lactone suicide substrate inactivates group VIA phospholipase A₂ by generating a diffiusible bromomethyl keto acid 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 PLA ₂. Group VIA PLA₂ (iPLA₂β) has a GTSTG serine lipase consensus sequence, and studies with a bromoenol lactone (BEL) suicide substrate inhibitor have been taken to suggest that iPLA ₂β participates in a wide variety of biological processes. Such conclusions presume inhibitor specificity. Inhibition by BEL requires its hydrolysis by and results in uncharacterized covalent modification(s) of iPLA₂β. We performed mass spectrometric analyses of proteolytic digests of BEL-treated iPLA₂β 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 iPLA ₂β Cys residues are alkylated at various BEL concentrations to form a thioether linkage to a BEL keto acid hydrolysis product. Synthetic Cys-containing peptides are alkylated when incubated with iPLA₂β and BEL, which reflects iPLA₂β-catalyzed BEL hydrolysis to a diffusible bromomethyl keto acid product that reacts with distant thiols. The BEL concentration dependence of Cys⁶⁵¹ alkylation closely parallels that of loss of iPLA₂β activity. No amino acid residues other than Cys were found to be modified, suggesting that Cys alkylation is the covalent modification of iPLA₂β responsible for loss of activity, and the alkylating species appears to be a diffusible hydrolysis product of BEL rather than a tethered acyl-enzyme intermediate.