A BROMOENOL LACTONE SUICIDE SUBSTRATE INACTIVATES GROUP VIA PHOSPHOLIPASE A2

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 磷脂酶A2（PLA 2）包括将磷脂水解成游离脂肪酸的酶超家族，例如，花生四烯酸和α 2-溶血磷脂。剖析它们各自的功能在很大程度上依赖于区分PLA 2的药理学抑制剂。VIA组PLA 2（iPLA 2）具有GTSTG丝氨酸脂肪酶共有序列，并且已经采用溴烯醇内酯（BEL）自杀底物抑制剂的研究表明iPLA 2参与多种生物过程。这样的结论假定抑制剂特异性。BEL的抑制需要其被iPLA 2水解并导致iPLA 2的未表征的共价修饰。我们对BEL处理的iPLA 2的蛋白水解酶进行质谱分析，以鉴定与活性丧失相关的修饰。GTSTG活性位点和序列的大侧翼区不被BEL处理修饰，但大多数iPLA 2 Cys残基在各种BEL浓度下被烷基化以形成与BEL酮酸水解产物的硫醚键。合成的含Cys的肽在与iPLA 2和BEL一起孵育时被烷基化，这反映了iPLA 2催化的BEL水解成可扩散的溴甲基酮酸产物，该产物与远位硫醇反应。Cys 651烷基化的BEL浓度依赖性与iPLA 2活性丧失的BEL浓度依赖性密切平行。未发现除Cys以外的氨基酸残基被修饰，表明Cys烷基化是iPLA 2的共价修饰，导致活性丧失，并且烷基化物质似乎是BEL的可扩散水解产物，而不是系留的酰基酶中间体。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Phospholipases A2 (PLA2) comprise a superfamily of enzymes that hydrolyze phospholipids to a free fatty acid, e.g., arachidonate, and a 2-lysophospholipid. Dissecting their individual functions has relied in large part on pharmacological inhibitors that discriminate among PLA2. Group VIA PLA2 (iPLA2) has a GTSTG serine lipase consensus sequence, and studies with a bromoenol lactone (BEL) suicide substrate inhibitor have been taken to suggest that iPLA2 participates in a wide variety of biological processes. Such conclusions presume inhibitor specificity. Inhibition by BEL requires its hydrolysis by and results in uncharacterized covalent modification(s) of iPLA2. We performed mass spectrometric analyses of proteolytic digests of BEL-treated iPLA2 to identify modifications associated with loss of activity. The GTSTG active site and large flanking regions of sequence are not modified by BEL treatment, but most iPLA2 Cys residues are alkylated at various BEL concentrations to form a thioether linkage to a BEL keto acid hydrolysis product. Synthetic Cys-containing peptides are alkylated when incubated with iPLA2 and BEL, which reflects iPLA2-catalyzed BEL hydrolysis to a diffusible bromomethyl keto acid product that reacts with distant thiols. The BEL concentration dependence of Cys651 alkylation closely parallels that of loss of iPLA2 activity. No amino acid residues other than Cys were found to be modified, suggesting that Cys alkylation is the covalent modification of iPLA2 responsible for loss of activity, and the alkylating species appears to be a diffusible hydrolysis product of BEL rather than a tethered acyl-enzyme intermediate.