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http://140.128.103.80:8080/handle/310901/23294
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| Title: | A phospholipid substrate molecule residing in the membrane surface mediates opening of the lid region in group IVA cytosolic phospholipase A2 |
| Authors: | Burke, J.E.ab, Hsu, Y.-H.ab, Deems, R.A.ab, Li, S.c, Woods Jr., V.L.c , Dennis, E.A.ab |
| Contributors: | Department of Chemistry, Tunghai University |
| Date: | 2008 |
| Issue Date: | 2013-06-11T09:06:15Z (UTC)
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| Abstract: | The Group IVA (GIVA) phospholipase A2 associates with natural membranes in response to an increase in intracellular Ca2+ along with increases in certain lipid mediators. This enzyme associates with the membrane surface as well as binding a single phospholipid molecule in the active site for catalysis. Employing deuterium exchange mass spectrometry, we have identified the regions of the protein binding the lipid surface and conformational changes upon a single phospholipid binding in the absence of a lipid surface. Experiments were carried out using natural palmitoyl arachidonyl phosphatidylcholine vesicles with the intact GIVA enzyme as well as the isolated C2 and catalytic domains. Lipid binding produced changes in deuterium exchange in eight different regions of the protein. The regions with decreased exchange included Ca2+ binding loop one, which has been proposed to penetrate the membrane surface, and a charged patch of residues, which may be important in interacting with the polar head groups of phospholipids. The regions with an increase in exchange are all located either in the hydrophobic core underneath the lid region or near the lid and hinge regions from 403 to 457. Using the GIVA phospholipase A2 irreversible inhibitor methyl-arachidonyl fluorophosphonate, we were able to isolate structural changes caused only by pseudo-substrate binding. This produced results that were very similar to natural lipid binding in the presence of a lipid interface with the exception of the C2 domain and region 466-470. This implies that most of the changes seen in the catalytic domain are due to a substrate-mediated, not interface-mediated, lid opening, which exposes the active site to water. Finally experiments carried out with inhibitor plus phospholipid vesicles showed decreases at the C2 domain as well as charged residues on the putative membrane binding surface of the catalytic domain revealing the binding sites of the enzyme to the lipid surface. ? 2008 by The American Society for Biochemistry and Molecular Biology, Inc. |
| Relation: | Journal of Biological Chemistry
Volume 283, Issue 45, 7 November 2008, Pages 31227-31236 |
| Appears in Collections: | [化學系所] 期刊論文
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