Interface Quality Effects in Phospholipase Membrane Enzymology

描述（由申请人提供）：磷脂酶是消化酶和外周膜酶，在膜-水界面催化磷脂水解。有充分的文件证据，在数据驱动的相关性的形式，界面的物理化学性质发挥了重要作用的水解速率。本计画的目标是建立一个范例，以解决磷脂酶膜酶学中尚未解决的界面质量效应问题。基于近年来对磷脂酶在胶束界面上的活性的研究，提出了磷脂酶在胶束界面上的动力学模型和界面的特殊功能：1）酶与囊泡结合形成E ~*，其平衡结合常数为KS;（2）E* 在活性位点与脂质结合形成界面复合物E*L，其结合和解离速率常数分别为k2和k-2;（3）脂类水解，速率常数为k3。具体地，E*L的缔合和解离是分别具有能量势垒5L和5 R的热活化过程，使得k2 = k20 exp（-5L/kBTB）和k-2 = k-20 exp（-5R/kBTB），其中kBB是玻尔兹曼常数。膜结构定义了能量δ L和δ R;因此速率常数k2和k-2;表面结合常数KS和k3（通过双层水合）。脂质类型和组成决定膜结构。因此，动力学参数KS、k2、k-2和k3是组成依赖性的。因此，界面作用的机理细节起源于E*L、E* 和水合作用的膜结构依赖性。目标是：1.开发一种新的磷脂酶动力学测定方法，采用底物L-磷脂及其非水解D-对映体以各种比例混合，以设计表面稀释系列。这样的混合物是一个长期存在的问题的解决方案的能力，以改变在双层界面基板浓度。通过完善的pH-stat以及采用FRET（荧光共振能量转移）荧光团标记的磷脂的新荧光测定法测量活性与底物浓度。将所提出的动力学方案得到的模型与数据拟合，并获得动力学参数。2.确定k 2和k-2的Arhenius温度依赖性的影响。用新型微量热法对配合物E*L进行了独立表征，得到了E*L的生成自由能。检查微量热数据和动力学数据之间的一致性。通过电子自旋共振测量双层水合，以确定与k3的相关性和对k3的影响。这项工作的意义是它的潜力，阐明术语“界面质量效应”通过新的范式，界面的物理化学性质的调节作用，通过动力学参数表示。这对人类健康很重要，因为水解产物执行几种生理功能，包括细胞信号传导、炎症、过敏、凋亡和肿瘤发生。 公共卫生相关性：脂解活性涉及一系列正常生理以及病理生理细胞功能，包括细胞信号传导、炎症、变态反应、凋亡和肿瘤发生。阐明膜的物理化学性质调节脂解活性的机制将为理解和最终控制健康和疾病中的这些生物事件提供重要信息。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.