INTERFACIAL ACTIVATION OF WATER-SOLUBLE PHOSPHOLIPASES

拟议的工作旨在了解复杂的界面行为 三种水溶性脂肪分解酶：细菌非特异性 磷脂酶C（PLC）和磷脂酰肌醇特异性磷脂酶C （PI-PLC），以及细菌和卷心菜磷脂酶D（PLD）。NMR，QLS， SANS和荧光技术与动力学结合使用。 分析以研究磷脂底物、类似物和抑制剂 与每种酶的相互作用一般性问题包括：（一）如何 磷脂界面在不同的聚集体结构中变化，并且可以 这些与对磷脂酶的敏感性有关吗？(ii)怎么 磷脂酶作用第二信使亲脂产物影响 基质聚集体？它们能潜在地调节磷脂酶吗？ (iii)磷脂与这些分子结合的关键参数是什么 酶-从磷脂结构的角度来看， 酶活性位点？(iv)我们能不能根据 这些结果？(v)能否建立一个统一的模型， 磷脂酶对作为单体、胶束、去污剂的底物的活性 混合胶束和双层囊泡 具体目标包括：（1）确定合成的 与PLC和PI-PLC结合的底物类似物;拟定PLC的检测 三角双锥反应中间体， （2）底物头基解析 使用充分表征的短链单体与PLC相互作用， 胶束和双层底物系统的动力学;克隆PLC Glu-4的位点特异性突变表明与胆碱相互作用 （3）钒酸盐对PLC的抑制作用的表征 用于提出的环状磷酸二酯中间体的模型;（4）确定 衬底横向和垂直扩散对PLC、PI的重要性， PLC和PLD与可聚合磷脂;（5）合成和 肌醇C2'和C6'修饰的短链PI的表征， 定义环如何与PI-PLC相互作用;和（6）使用荧光- 标记的DAG以监测不同磷脂之间的混合和转移 聚集体和PA的表征PLD的抑制和PA的作用 不同的脂质聚集体。 这些研究的结果应能使人们更好地了解 这些磷脂酶的反应机制以及产物， 特别是脂质第二信使，和底物物理 特性影响磷脂酶活性。由于这些脂质第二 信使激活细胞生长，它们与广泛的 疾病以及正常的细胞功能。

The proposed work seeks to understand the complex interfacial behavior of three water-soluble lipolytic enzymes: bacterial nonspecific phospholipase C (PLC) and phosphatidylinositol-specific phospholipase C (PI-PLC), and bacterial and cabbage phospholipase D (PLD). NMR, QLS, SANS, and fluorescence techniques are used in conjunction with kinetic analyses to investigate phospholipid substrate, analog, and inhibitor interactions with each enzyme. General questions include: (i) How do phospholipid interfaces vary in different aggregate structures, and can these be correlated with susceptibility to phospholipases? (ii) How do the second messenger lipophilic products of phospholipase action affect the substrate aggregate? Can they potentially regulate phospholipases? (iii) What are the critical parameters for phospholipid binding to these enzymes - both from the point of view of phospholipid structure and enzyme active sites? (iv) Can we design specific inhibitors based on these results? (v) Can a unified model be developed that accounts for phospholipase activity toward substrate as monomer, micelles, detergent mixed micelles, and bilayer vesicles? Specific aims include: (1) Determination of the conformation of synthetic substrate analogs bound to PLC and PI-PLC; detection of the proposed PLC trigonal bipyramidal reaction intermediate using time-resolved solid- state 31P NMR techniques; (2) Elucidation of substrate headgroup interactions with PLC using well-characterized short-chain monomer, micellar, and bilayer substrate systems for kinetics; cloning PLC for site-specific mutagenesis of Glu-4 suggested to interact with the choline N(CH3)3 moiety; (3) Characterization of vanadate inhibition of PLC as a model for a proposed cyclic phosphodiester intermediate; (4) Determining the importance of substrate lateral and vertical diffusion for PLC, PI- PLC, and PLD with polymerizable phospholipids; (5) Synthesis and characterization of inositol C2' and C6' modified short-chain PI's to define how the ring interacts with PI-PLC; and (6) Use of fluorescent- labeled DAGs to monitor mixing and transfer among different phospholipid aggregates and characterization of PA inhibition of PLD and effect of PA on different lipid aggregates. The results of these studies should provide a better understanding of the reaction mechanisms for these phospholipases and how products, specifically lipid second messengers, and substrate physical characteristics affect phospholipase activity. Since these lipid second messengers activate cell growth, they have relevance to a wide range of diseases as well as to normal cell functions.