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.
|Number of pages||13|
|State||Published - Jan 24 2006|