Highly selective hydrolysis of fatty acyl-CoAs by calcium-independent phospholipase A2β -: Enzyme autoacylation and acyl-CoA-mediated reversal of calmodulin inhibition of phospholipase A2 activity
Highly selective hydrolysis of fatty acyl-CoAs by calcium-independent phospholipase A2β -: Enzyme autoacylation and acyl-CoA-mediated reversal of calmodulin inhibition of phospholipase A2 activity
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DOI：
10.1074/jbc.m511623200
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发表时间：
2006-06-09
影响因子：
4.8
通讯作者：
Gross, Richard W.
中科院分区：
文献类型：
--
作者：
Jenkins, Christopher M.;Yan, Wei;Gross, Richard W.
Calcium-independent phospholipase A(2)beta (iPLA(2)beta) participates in numerous diverse cellular processes, such as arachidonic acid release, insulin secretion, calcium signaling, and apoptosis. Herein, we demonstrate the highly selective iPLA(2)beta-catalyzed hydrolysis of saturated long-chain fatty acyl-CoAs (palmitoyl-CoA approximate to myristoyl-CoA "stearoyl-CoA" oleoyl-CoA approximate to arachidonoyl-CoA) present either as monomers in solution or guests in host membrane bilayers. Site-directed mutagenesis of the iPLA(2)beta catalytic serine (S465A) completely abolished acyl-CoA thioesterase activity, demonstrating that Ser-465 catalyzes both phospholipid and acyl-CoA hydrolysis. Remarkably, incubation of iPLA(2)beta with oleoyl-CoA, but not other long-chain acyl-CoAs, resulted in robust stoichiometric covalent acylation of the enzyme. Moreover, S465A mutagenesis or pretreatment of wild-type iPLA(2)beta with (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2- one unexpectedly increased acylation of the enzyme, indicating the presence of a second reactive nucleophilic residue that participates in the formation of the fatty acyl-iPLA(2)beta adduct. Radiolabeling of intact Sf9 cells expressing iPLA(2)beta with [H-3] oleic acid demonstrated oleoylation of the membrane-associated enzyme. Partial trypsinolysis of oleoylated iPLA(2)beta and matrix-assisted laser desorption ionization mass spectrometry analysis localized the acylation site to a hydrophobic 25-kDa fragment (residues similar to 400-600) spanning the active site to the calmodulin binding domain. Intriguingly, calmodulin-Ca2+ blocked acylation of iPLA(2)beta by oleoyl-CoA. Remarkably, the addition of low micromolar concentrations ( 5 mu M) of oleoyl-CoA resulted in reversal of calmodulin-mediated inhibition of iPLA(2)beta phospholipase A(2) activity. These results collectively identify the molecular species-specific acyl-CoA thioesterase activity of iPLA(2)beta, demonstrate the presence of a second active site that mediates iPLA(2)beta autoacylation, and identify long-chain acyl-CoAs as potential candidates mediating calcium influx factor activity.
