Mechanism-based inhibition of iPLA2ß demonstrates a highly reactive cysteine residue (C651) that interacts with the active site: mass spectrometric elucidation of the mechanisms underlying inhibition.
Biochemistry
; 52(24): 4250-63, 2013 Jun 18.
Article
in En
| MEDLINE
| ID: mdl-23701211
ABSTRACT
The multifaceted roles of calcium-independent phospholipase A2ß (iPLA2ß) 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 iPLA2ß 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 iPLA2ß constructed from the crystal structure of iPLA2α (patatin) indicate that the distance between S465 and C651 is approximately 10 Å within the active site of iPLA2ß. Kinetic analysis of the formation of the 75 kDa iPLA2ß-BEL species with the (R) and (S) enantiomers of BEL demonstrated that the reaction of (S)-BEL with iPLA2ß was more rapid than for (R)-BEL paralleling the enantioselectivity for the inhibition of catalysis by each inhibitor with iPLA2ß. Moreover, we demonstrate that the previously identified selective acylation of iPLA2ß 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 iPLA2ß 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 iPLA2ß and the topology of the active site.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Cysteine
/
Phospholipases A2, Calcium-Independent
Limits:
Animals
Language:
En
Journal:
Biochemistry
Year:
2013
Document type:
Article
Affiliation country: