Interfacial Catalysis by Phospholipase A2

描述：（申请人的描述）许多酶，包括脂质的那些酶， 代谢，已经进化到在界面处访问它们的底物，并且 在胶束的界面处进行催化翻转，或者 双层膜例如，中有十几种不同的磷脂酶A2（PLA21） 人体组织作用于膜的磷脂。产品及其 类二十烷酸代谢物发出信号并调节宽范围的信号， 分泌和炎症过程。我们已经制定了 PLA 2界面动力学分析挑战在于剖析 来自其他过程的界面催化转换事件， 整体平均的微观稳态条件。解剖 酶从界面催化剂结合到界面， 周转事件允许确定基本利率，均衡， 激活参数这种方法导致了对 催化机理界面活化被分解为增加的底物 酶在界面处的亲和力（Ksstar-变构），和kcatstar 活化由阴离子界面介导。 我们的下一个重点是发展变构界面的结构基础 activation.我们的工作模型是，胰腺PLA2在界面存在， 以两种形式存在：在阴离子界面处的电荷补偿形式是 催化活性（EstarS）#，和电荷敏感形式， 两性离子界面（EstarS）受损（方案II）。的具体目标 提出的研究包括：（#1）辨别Lys-10和Lys-62的作用和（#2） PLA2中的63 - 66环与界面结合， 事件在1，10，19，20，31，53，56，62，69，73，87， 115或120将使用W3F以及K53、56、121 M（含或不含）进行制备 替代来表示电荷敏感和电荷补偿形式 的猪胰腺PLA2（同种型IB）。Trp突变体的两种形式将是 特征在于（#3）催化、活化和结合参数， 阴离子与两性离子界面，以及（#4）光谱学上， 确定在不同条件下单个Trp探针的猝灭剂可及性 岗位目标1 - 4的结果将用于确定 与界面接触的IB PLA2（i面），以及该面如何 在阴离子与两性离子界面处不同。（#5）阴离子结合 从共晶的X射线结构中可以确定PLA2的位置 某些阴离子，如硫酸根和磷酸根。（#6）主要结果 目标将扩展到其他PLA2亚型及其定点突变体。 因此，总体目标是识别i-面处的残基，识别i-面处的残基， 阴离子结合位点，表征三级结构和功能 PLA2亚型之间的差异。这些结果将提供洞察力， 的耦合之间的功能差异的结构基础 i-面与PLA2家族的活性位点事件。

DESCRIPTION: (Applicant's Description) Many enzymes, including those of lipid metabolism, have evolved to access their substrates at the interface, and processively carry out the catalytic turnover at the interfaces of micelles or bilayer membrane. For example, a dozen different phospholipase A2 (PLA21) in human tissues act on phospholipids of membranes. The products and their eicosanoid metabolites signal and regulate a wide range of signaling, secretory, and inflammatory processes. We have established protocols for the interfacial kinetic analysis of PLA2. The challenge is to dissect the interfacial catalytic turnover events from other processes that influence the microscopic steady state condition for the ensemble-averaging. Dissection of the binding of the enzyme to the interface from the interfacial catalytic turnover events permits determination of the primary rate, equilibrium, and activation parameters. This approach has led to novel insights into the catalytic mechanism. Interfacial activation is dissected as increased substrate affinity of the enzyme at the interface (Ksstar-allostery), and the kcatstar activation is mediated by the anionic interface. Our next focus is to develop a structural basis for the allosteric interfacial activation. Our working model is that pancreatic PLA2 at the interface exists in two forms: the charge-compensated form at the anionic interface is catalytically active (EstarS)#, and the charge-sensitive form at the zwitterionic interface (EstarS) is impaired (Scheme II). Specific aims of the proposed study include: (#1) To discern the role of Lys-l0 and Lys-62 and (#2) of the 63-66 loop in the PLA2 binding to the interface and the catalytic events. Mutants with a single Trp at 1, 10, 19,20, 31, 53, 56, 62 ,69, 73, 87, 115 or 120 will be prepared with the W3F and with or without the K53,56, 121M substitution to represent the charge-sensitive and the charge-compensated forms of pig pancreatic PLA2 (isoform IB). Both forms of the Trp-mutants will be characterized for (#3) the catalytic, activation, and binding parameters at the anionic versus zwitterionic interfaces, and also (#4) spectroscopically to ascertain the quencher accessibility of the single Trp-probe in different positions. Together, results of aims #1-4 will be used to identify the face of IB PLA2 (the i-face) that makes contact with the interface, and how this face differs at the anionic versus zwitterionic interfaces. (#5) The anion binding sites of PLA2 will be identified from the x-ray structure of the cocrystals with certain anions, such as sulfate and phosphate. (#6) Key results from these aims will be extended to other PLA2 isoforms and their site-directed mutant's. Thus the overall goal is to identify the residues at the i-face, identify the anion binding sites, characterize the tertiary structural and functional differences between the PLA2 isoforms. These results will provide insights into the structural basis for the functional differences between the coupling of the i-face with the active site events of the PLA2 family.