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Interactions of cytosolic phospholipase A2 with pyrrophenone, ca2+ and phospholipids studied using deuterium exchange mass spectrometry
Author(s) -
Burke John E,
Hsu Howard,
Deems Raymond,
Woods Virgil,
Dennis Edward A
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.611.21
Subject(s) - chemistry , hydrogen–deuterium exchange , phospholipid , cytosol , phospholipase , phospholipase a , amide , phospholipase a2 , enzyme , membrane , arachidonic acid , biochemistry , biophysics , mass spectrometry , chromatography , biology
Phospholipase A 2 (PLA 2 ) catalyzes hydrolysis of the sn ‐2 fatty acyl bond of phospholipids (PL), liberating free fatty acid and lysophospholipid. We are studying the)mechanism of activation, and membrane binding of the cytosolic GIVA PLA 2 . This enzyme liberates arachidonic acid from phospholipids, which is a precursor formany inflammatory mediators. We have used hydrogen‐deuterium exchange mass spectrometry (DXMS) to study this enzyme. The rate of exchange of each amide proton is directly proportional to the accessibility to bulk solvent. Measurements of exchange rates have been used to characterize changes in solvent accessibility that result from protein/lipid binding interactions, and conformational changes. Group IVA cPLA 2 's interactions with inhibitors and phospholipid were studied using DXMS. We used pyrrophenone, a potent inhibitor of GIVA PLA 2 , to probe the changes in conformation upon binding in the active site. Large amide exchange rate changes were seen upon inhibitor binding. We propose that these changes can be explained by the opening of the lid region with inhibitor bound. Large changes were also seen upon calcium‐mediated lipid binding, mainly in the C2 domain. We propose the protein inserting into the lipid membrane causes these changes. These studies show that DXMS is an excellent technique for probing enzyme interfacial activation. This work was supported by NIHGM20,501