A BROMOENOL LACTONE SUICIDE SUBSTRATE INACTIVATES GROUP VIA PHOSPHOLIPASE A2

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 磷脂酶A2(PLA2)是一个超家族的酶，能将磷脂分解成游离脂肪酸，如花生四烯酸和2-溶血磷脂。剖析它们的个体功能在很大程度上依赖于区分PLA2的药理抑制剂。通过磷脂酶A2的基团(IPLA2)具有GTSTG丝氨酸脂酶的共同序列，而对溴烯醇内酯(BEL)自杀底物抑制剂的研究表明，iPLA2参与了广泛的生物过程。这些结论假定了抑制剂的专一性。BEL的抑制需要iPLA2的水解和未表征的共价修饰(S)的结果。我们对BEL处理的iPLA2的蛋白水解酶进行了质谱分析，以确定与活性丧失相关的修饰。BEL处理不会改变GTSTG活性部位和序列的大侧翼区域，但大多数iPLA2半胱氨酸残基在不同BEL浓度下被烷基化，形成与BEL酮酸水解产物的硫醚键。合成的含半胱氨酸的多肽在与iPLA2和BEL孵育时发生烷基化，这反映了iPLA2催化的BEL水解为可扩散的溴甲基酮酸产物，该产物可与远处的硫醇反应。Cys651烷基化的BEL浓度依赖关系与iPLA2活性的丧失密切相关。没有发现除半胱氨酸以外的氨基酸残基被修饰，这表明半胱氨酸烷基化是导致活性丧失的iPLA2的共价修饰，烷基化物种似乎是BEL的扩散性水解产物，而不是连接的酰基酶中间体。

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.