Effects of biological oxidants on the catalytic activity and structure of group VIA phospholipase A₂

Group VIA phospholipase A₂ (iPLA₂β) is expressed in phagocytes, vascular cells, pancreatic islet β-cells, neurons, and other cells and plays roles in transcriptional regulation, cell proliferation, apoptosis, secretion, and other events. A bromoenol lactone (BEL) suicide substrate used to study iPLA₂β functions inactivates iPLA ₂β by alkylating Cys thiols. Because thiol redox reactions are important in signaling and some cells that express iPLA₂β produce biological oxidants, iPLA₂β might be subject to redox regulation. We report that biological concentrations of H₂O ₂, NO, and HOCl inactivate iPLA₂β, and this can be partially reversed by dithiothreitol (DTT). Oxidant-treated iPLA ₂β modifications were studied by LC-MS/MS analyses of tryptic digests and included DTT-reversible events, e.g., formation of disulfide bonds and sulfenic acids, and others not so reversed, e.g., formation of sulfonic acids, Trp oxides, and Met sulfoxides. W⁴⁶⁰ oxidation could cause irreversible inactivation because it is near the lipase consensus sequence (⁴⁶³GTSTG⁴⁶⁷), and site-directed mutagenesis of W ⁴⁶⁰ yields active mutant enzymes that exhibit no DTT-irreversible oxidative inactivation. Cys651-sulfenic acid formation could be one DTT-reversible inactivation event because Cys651 modification correlates closely with activity loss and its mutagenesis reduces sensitivity to inhibition. Intermolecular disulfide bond formation might also cause reversible inactivation because oxidant-treated iPLA₂β contains DTT-reducible oligomers, and oligomerization occurs with time- and temperature-dependent iPLA₂β inactivation that is attenuated by DTT or ATP. Subjecting insulinoma cells to oxidative stress induces iPLA₂β oligomerization, loss of activity, and subcellular redistribution and reduces the rate of release of arachidonate from phospholipids. These findings raise the possibility that redox reactions affect iPLA₂β functions.