Mechanism-Based Inhibition of iPLA(2)beta Demonstrates a Highly Reactive Cysteine Residue (C651) That Interacts with the Active Site: Mass Spectrometric Elucidation of the Mechanisms Underlying Inhibition

作者：Jenkins Christopher M; Yang Jingyue; Gross Richard W^*

来源：Biochemistry, 2013, 52(24): 4250-4263.

DOI：10.1021/bi4004233

The multifaceted roles of calcium-independent phospholipase A(2)beta (iPLA(2)beta) in numerous cellular processes have been extensively examined through utilization of the iPLA(2)-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(2)beta 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(2)beta constructed from the crystal structure of iPLA(2)alpha (patatin) indicate that the distance between S465 and C651 is approximately 10 angstrom within the active site of iPLA(2)beta. Kinetic analysis of the formation of the 75 kDa iPLA(2)beta-BEL species with the (R) and (S) enantiomers of BEL demonstrated that the reaction of (S)-BEL with iPLA(2)beta was more rapid than for (R)-BEL paralleling the enantioselectivity for the inhibition of catalysis by each inhibitor with iPLA(2)beta. Moreover, we demonstrate that the previously identified selective acylation of iPLA(2)beta 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 iPLA(2)beta 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 iPLA(2)beta and the topology of the active site.

• 出版日期2013-6-18