BROMOENOL LACTONE SUICIDE SUBSTR INACTIVATES IPLA2B BY GENERATING A DIFF

溴烯醇内酯自杀底物通过产生差异使 IPLA2B 失活

• 批准号: 7355273
• 负责人: HAOWEI SONG
• 金额: $ 0.85万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7355273
• 关键词: BROMOENOL; LACTONE; SUICIDE; SUBSTR; INACTIVATES

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. A bromoenol lactone suicide substrate inactivates Group VIA phospholipase A2 by generating a diffusible bromomethyl ketoacid that alkylates cysteine thiols: 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 suggest that iPLA2¿ participates in a wide variety of biological processes. 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] to form a thioether linkage to a BEL ketoacid hydrolysis product. Synthetic Cys-containing peptides are alkylated when incubated with iPLA2¿ and BEL, which reflects iPLA2¿-catalyzed BEL hydrolysis to a diffusible bromomethyl-ketoacid product that reacts with distant thiols. Cys651 alkylation is strongly associated with loss of iPLA2b activity, and no amino acid residues other than Cys were found to be modified, suggesting that Cys651 might play a previously unsuspected role in catalysis, substrate recognition, or maintaining an active conformation of iPLA2¿.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。溴烯醇内酯自杀底物通过产生烷基化半胱氨酸硫醇的可扩散的溴甲基酮酸使VIA族磷脂酶A2失活：磷脂酶A2（PLA 2）包含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 PLA 2（iPLA 2）组）具有GTSTG丝氨酸脂肪酶共有序列，并且用溴烯醇内酯（BEL）自杀底物抑制剂的研究表明iPLA 2 <$参与多种生物过程。BEL的抑制作用需要其水解，并导致iPLA 2的未表征的共价修饰。我们对BEL处理的iPLA 2？的蛋白水解酶进行了质谱分析，以鉴定与活性丧失相关的修饰。GTSTG活性位点和序列的大侧翼区不被BEL处理修饰，但大多数iPLA 2 <$Cys残基在不同的[BEL]处被烷基化，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水解为可扩散的溴甲基-酮酸产物，该产物与远端硫醇反应。Cys 651烷基化与iPLA 2b活性的丧失密切相关，并且没有发现Cys以外的氨基酸残基被修饰，这表明Cys 651可能在催化、底物识别或维持iPLA 2？的活性构象方面发挥先前未被怀疑的作用。