Phospholipases are digestive as well as peripheral membrane enzymes that catalyze phospholipid hydrolysis at the membrane-water interface. There is well-document evidence, in the form of data driven correlations only, that interface physicochemical properties play a major role in the rate of hydrolysis. The goal of this project is to establish a paradigm for the yet unsolved problem of the interface quality effects in phospholipase membrane enzymology. Based on recently concluded work on phospholipase activity at micellar interfaces, a kinetic scheme and specific function of the interface for bilayers are hypothesized as follows: The three key sequential steps are: 1) enzyme- binds to vesicle to form E* with equilibrium binding constant KS,;2) E* binds lipid at the active site to form the interfacial complex E*L with association and dissociation rate constants k2 and k-2 respectively;and 3) lipid hydrolysis with rate constant k3. Specifically, the association and dissociation of E*L are thermally activated processes with energy barriers 5L and 5R respectively, so that k2 = k20 exp (-5L/kBTB ) and k-2 = k-20 exp (-5R/kBTB ), where kBB is the Boltzmann constant. Membrane structure defines the energies 5L and 5R;and thus the rate constants k2 and k-2;the surface binding constant, KS and k3 (via bilayer hydration). Lipid type and composition define membrane structure. Hence the kinetic parameters KS, k2, k-2, and k3 are composition dependent. Thus the mechanistic details of the role of the interface originates in the membrane-structure dependent properties of E*L, E*, and hydration.
The aims are: 1. Develop a novel assay for phospholipase kinetics employing mixtures of the substrate L-phospholipids and their non- hydrolyzing D-enantiomers in various proportions to design a surface dilution series. Such a mixture is a solution to a long-standing problem of the ability to vary the interface substrate concentration in bilayers. Measure activity vs. substrate concentration, by the well established pH-stat as well as new fluorogenic assays employing phospholipids labeled with FRET (fluorescence resonance energy transfer) fluorophores. Fit the model resulting from the proposed kinetic scheme to the data and obtain the kinetic parameters. 2. Determine the effects of the Arhenius temperature dependence of k2 and k-2. Characterize the complex E*L independently by novel microcalorimetry and obtain the free energy of formation of E*L. Examine the agreement between the microcalorimetry data and the kinetic data. Measure bilayer hydration by Electron Spin Resonance to determine correlation with and effect on k3. The significance of this work is its potential to elucidate the term """"""""interface quality effects"""""""" through the new paradigm that the regulatory role of the interface physicochemical properties is expressed through the kinetic parameters. This is of importance to human health because the products of hydrolysis perform several physiological functions including cell signaling, inflammation, allergy, apoptosis, and tumorigenesis.
Lipolytic activity is involved in a series of normal physiological as well as pathophysiological cell functions including cell signaling, inflammation, allergy, apoptosis and tumorigenesis. Elucidation of the mechanism by which membrane physicochemical properties regulate lipolytic activity will provide important information toward understanding and eventually controlling these biological events in health and disease.
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