INTERFACIAL CATALYSIS BY PHOSPHOLIPASE A2

有几个过程是由界面催化调节的， 膜表面 因此，磷脂酶A2（PLA 2）是令人感兴趣的 不仅作为界面催化的原型， 还因为它们动员了生物合成的前体 类花生酸和血小板活化因子， 控制广泛的分泌和炎症过程。 的 界面催化研究的长期目标是 获得详细的动力学，生物物理学和分子知识， PLA 2在接口处。 对该机制的理解将 也有助于设计，分析和表征特定的 抑制剂，这可能是有用的建立和控制 PLA 2的生物学功能。 在接下来的五年里，我们计划利用动能和 本实验室最近发展的平衡法 PLA 2（I型）的目的是实现以下目标： 其他PLA 2的平衡和动力学参数，例如 从滑液中克隆的II型酶， 蜜蜂毒液，和花生四烯酸特异性89 KD酶从蜜蜂毒液， （B）表征所述氨基酸 暴露于水相的酶上的残基结合到 （c）光谱表征E、E* 和E*L （d）确定催化剂或产物是否 释放步骤在催化转换期间是限速的;（e） 研究短链底物水解的动力学 在它们作为单独单体分散的条件下， 以获得洞察力的可能性形成的 一个孤立的ES复合物;（f）表征的动力学 与胆汁盐共分散的长链磷脂的水解 以检查在小聚集体上水解速率是否受限 由基质的补充速率决定。 通过整合 从这些研究中获得的信息，我们希望获得一个普遍的 和完整的动力学描述的界面催化的PLA 2 从几个来源。 这些研究将提供物理和 分子的洞察力，不仅发生在过程中， 酶与界面的结合，还有那些 抑制剂和产物在界面处与PLA 2的结合。 PLA 2的特定突变体也将用于鉴定残基 参与酶与界面的结合， 参与配体与催化活性物质结合的残基 站点区域。

Several processes are regulated by interfacial catalysis on the membrane surfaces. Thus phospholipase A2 (PLA2) are interesting not only as prototypes for interfacial catalysis in general, but also because they mobilize precursors for the biosynthesis of eicosanoids and platelet activating factor, which ultimately control a wide range of secretory and inflammatory processes. The long range objective of our studies on interfacial catalysis is to gain detailed kinetic, biophysical, and molecular knowledge about PLA2 at the interface. An understanding of the mechanism would also help in the design, assay and characterization of specific inhibitors, which could be useful in establishing and controlling the biological functions of PLA2. During the next five years we plan to use the kinetic and equilibrium methods developed recently in our laboratory for pig PLA2 (type I) to achieve the following objectives: (a) to obtain the equilibrium and kinetic parameters for other PLA2, such as the cloned type II enzyme from synovial fluid, the type III PLA2 from bee venom, and the arachidonate-specific 89 KD enzyme from the cytoplasm of a human cell line; (b) to characterize the amino acid residues exposed to the aqueous phase on the enzyme bound to the interface; (c) to spectroscopically characterize the E, E*, and E*L forms of PLA2; (d) to establish if the catalytic or the product release step is rate-limiting during the catalytic turnover; (e) to investigate the kinetics of hydrolysis of short chain substrates under the conditions where they are dispersed as solitary monomers, so as to obtain insights into the possibility of the formation of a solitary ES complex; (f) to characterize the kinetics of hydrolysis of long chain phospholipids codispersed with bile salts to examine if on small aggregates the rate of hydrolysis is limited by the rate of replenishment of the substrate. By integrating the information obtained from such studies, we hope to obtain a general and complete kinetic description of interfacial catalysis by PLA2 from several sources. These studies would provide physical and molecular insights into the processes that occur not only on the binding of the enzyme to the interface, but also those that occur on the binding of inhibitors and products to PLA2 at the interface. Specific mutants of PLA2 will also be used to identify the residues involved in the binding of the enzyme to the interface and the residues involved in the binding of ligands to the catalytic active site region.