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.
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