Mechanism-based inhibition of iPLA₂β demonstrates a highly reactive cysteine residue (C651) that interacts with the active site: Mass spectrometric elucidation of the mechanisms underlying inhibition

The multifaceted roles of calcium-independent phospholipase A ₂β (iPLA₂β) in numerous cellular processes have been extensively examined through utilization of the iPLA₂-selective inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL). Herein, we employed accurate mass/high resolution mass spectrometry to demonstrate that the active site serine (S465) and C651 of iPLA ₂β are covalently cross-linked during incubations with BEL demonstrating their close spatial proximity. This cross-link results in macroscopic alterations in enzyme molecular geometry evidenced by anomalous migration of the cross-linked enzyme by SDS-PAGE. Molecular models of iPLA ₂β constructed from the crystal structure of iPLA ₂α (patatin) indicate that the distance between S465 and C651 is approximately 10 Å within the active site of iPLA₂β. Kinetic analysis of the formation of the 75 kDa iPLA₂β-BEL species with the (R) and (S) enantiomers of BEL demonstrated that the reaction of (S)-BEL with iPLA₂β was more rapid than for (R)-BEL paralleling the enantioselectivity for the inhibition of catalysis by each inhibitor with iPLA₂β. Moreover, we demonstrate that the previously identified selective acylation of iPLA₂β by oleoyl-CoA occurs at C651 thereby indicating the importance of active site architecture for acylation of this enzyme. Collectively, these results identify C651 as a highly reactive nucleophilic residue within the active site of iPLA₂β which is thioesterified by BEL, acylated by oleoyl-CoA, and located in close spatial proximity to the catalytic serine thereby providing important chemical insights on the mechanisms through which BEL inhibits iPLA₂β and the topology of the active site.