INTERFACIAL CATALYSIS BY PHOSPHOLIPASE A2

描述：专门的酶已经进化为催化周转， 在水分散体中形成有序界面的天然基质。 其中，磷脂酶A2不仅作为 界面催化研究原型和膜活性模型 proteins. 它们还包括一类有前途的药理学靶点 因为它们控制广泛的分泌和炎症过程， 包括细胞防御机制 这些药物的药理学前景 目标尚未实现，主要是因为动力学协议 界面催化没有充分建立。 远程 目的是发展对界面催化的详细了解 和磷脂酶A2的激活，并在蛋白质中解释这一点 结构术语。 1991-1995年期间的重点是剖析 从界面中的催化转化步骤结合到界面。 这使得分析描述的高度进行性反应， 界面催化和平衡参数。 发展 界面结合与事件之间的结构-功能关系 的催化循环，假设是，结合分泌 磷脂酶A2的接口需要多个网站，一些 这些相互作用通过钙调节催化参数 辅因子 具体目标是：（a）确立阳离子的作用 （B）开发一种分析方法， 短链底物水解动力学的描述;（c）至 发展界面结合的动力学和结构基础， （d）表征干扰的抑制剂的作用 与酶结合的界面;和（e）映射的 脂质暴露区域，并确定接触界面的残基。 等 研究将扩展界面催化的动力学描述， 最终有助于描述 酶的活化形式。 动力学和平衡参数将 提供物理和分子相关的过程，发生在 酶与界面的结合，以及 钙、底物、抑制剂和产物在界面处与PLA 2结合。 这 信息是无法从可溶性形式的研究， 酶在水相中，或通过通常的方法， 确定大分子结构（X射线或核磁共振）;然而，它 为功能与结构特征的相关性提供了基础， 将通过使用合适的突变体进一步进行。 调查人员 还将继续寻找、开发和使用PLA 2的特异性抑制剂 因为它们对细胞过程的影响，这最终应该有助于 实现界面酶的药理学潜力。

DESCRIPTION: Specialized enzymes have evolved for the catalytic turnover of natural substrates which form organized interfaces in aqueous dispersions. Among these, the phospholipases A2 have attracted attention not only as prototypes for study of interfacial catalysis and models of membrane active proteins. They also comprise a class of promising pharmacological targets because they control a wide range of secretory and inflammatory processes, including cellular defense mechanisms. The pharmacological promise of these targets has not been realized yet, largely because the kinetic protocols for interfacial catalysis are not adequately established. The long-range objective is to develop a detailed understanding of interfacial catalysis and activation by phospholipase A2, and to account for this in protein structural terms. The focus during the 1991-1995 period was dissecting the binding to the interface from catalytic turnover steps in the interface. This allowed an analytical description of the highly processive reaction in terms of interfacial catalytic and equilibrium parameters. To develop structure-function relationships between the interfacial binding and events of the catalytic cycle, the hypothesis is that the binding of secreted phospholipase A2 to the interface requires multiple sites, and that some of these interactions modulate the catalytic parameters through the calcium cofactor. The specific goals are: (a) to establish the role of the cation on the substrate binding and chemical steps; (b) to develop an analytical description of the kinetics of hydrolysis of short chain substrates; (c) to develop a kinetic and structural basis of interfacial binding and activation; (d) to characterize the effects of inhibitors that interfere with the binding of enzyme to the interface; and (e) to map the lipid-exposed region and identify residues that contact the interface. Such studies will extend the kinetic description of interfacial catalysis and ultimately help in the characterization of the activated form of the enzyme. The kinetic and equilibrium parameters will provide physical and molecular correlates for the processes that occur on the binding of the enzyme to the interface, as well as on the binding of calcium, substrate, inhibitors and products to PLA2 at the interface. This information is not accessible from the study of the soluble form of the enzyme in the aqueous phase, or through the usual methods for the determination of macromolecular structure (X-ray or NMR); however, it provides a basis for correlation of function with structural features, which will be pursued further by the use of suitable mutants. The investigators will also continue to search, develop and use specific inhibitors of PLA2 for their effects on cellular processes, which should ultimately help in realizing the pharmacological potential of interfacial enzymes.