Interfacial Catalysis by Phospholipase A2

描述：(应聘者的描述)多种酶，包括脂类 新陈代谢，已经进化到在界面上接触它们的底物，并且 在胶束界面或界面上连续进行催化周转 双层膜。例如，十几种不同的磷脂酶A2(PLA21) 人体组织作用于膜上的磷脂。这些产品和他们的 二十烷类代谢物发出信号并调节广泛的信号， 分泌和炎症过程。我们已经制定了协议， PLA2的界面动力学分析。挑战在于剖析 来自其他过程的界面催化周转事件影响 集合平均的微观稳态条件。剖析 酶通过界面催化作用与界面的结合 周转事件允许确定基本汇率、均衡和 激活参数。这种方法带来了对 催化机理。界面活化作为增加的底物被解剖 酶在界面上的亲和力(Ksstar-变构)，以及kcatstar 活化是由阴离子界面介导的。 我们的下一个重点是开发变构界面的结构基础 激活。我们的工作模型是胰腺PLA2在界面上存在 有两种形式：阴离子界面的电荷补偿形式为 催化活性(EstarS)#和电荷敏感形式 两性离子界面(EstarS)受损(方案II)。《公约》的具体目标 建议的研究包括：(1)辨别Lys-10和Lys-62的作用；(2) 在PLA2结合到界面上的63-66环和催化 事件。在1、10、19、20、31、53、56、62、69、73、87处有一个Trp的突变体， 115或120将与W3F一起准备，并带有或不带有K53、56、121M 表示电荷敏感形式和电荷补偿形式的代换 猪胰腺磷脂酶A2(异构体IB)。两种形式的Trp突变体都将是 表征了(#3)的催化、活化和结合参数 阴离子与两性离子界面，以及(#4)光谱到 单根色氨酸探针在不同温度下淬火可及性的确定 各就各位。总而言之，AIMS#1-4的结果将用于识别人脸 IB PLA2(与接口接触的I-Face)，以及该Face如何 在阴离子和两性离子界面上有所不同。(#5)阴离子结合 PLA2的位置将从共晶体的x射线结构中确定 含有某些阴离子，如硫酸盐和磷酸盐。(#6)这些项目的主要结果 AIMS将扩展到其他PLA2亚型及其定点突变体。 因此，总体目标是鉴定I-Face处的残基，鉴定 阴离子结合部位，表征三级结构和功能 PLA2亚型之间的差异。这些结果将为我们提供关于 联轴器之间的功能差异的结构基础 I-面对PLA2家族的活跃站点事件。

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.