Interface Quality Effects in Phospholipase Membrane Enzymology

磷脂酶膜酶学中的界面质量效应

• 批准号: 8265613
• 负责人: Radha Ranganathan
• 金额: $ 10.88万
• 依托单位: CALIFORNIA STATE UNIVERSITY NORTHRIDGE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8265613
• 关键词: Active Sites; Agreement; Apoptosis; Bee Venoms; Binding; Binding Sites; Biological; Biological Assay; Cell physiology; Cleaved cell; Complex; Data; Dependence; Disease; Dissociation; Electron Spin Resonance Spectroscopy; Enzymatic Biochemistry; Enzymes; Equilibrium; Esters; Event; Family suidae; Fatty Acids; Fluorescence Resonance Energy Transfer; Free Energy; Goals; Health; Human; Hydration status; Hydrolysis; Hypersensitivity; Inflammation; Kinetics; Label; Lipid Binding; Lipids; Lipolysis; Measurement; Measures; Membrane; Membrane Lipids; Microscopic; Modeling; Mole the mammal; Outcome; Pancreas; Peripheral; Phospholipase; Phospholipase A2; Phospholipids; Physiological; Play; Process; Property; Reaction; Regulation; Role; Scheme; Series; Signal Transduction; Solutions; Surface; Temperature; Testing; Vesicle; Water; Work; base; design; enantiomer; fluorophore; innovation; interfacial; microcalorimetry; novel; tumorigenesis

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.

描述（由申请人提供）：磷脂酶是一种消化酶和外周膜酶，在膜-水界面催化磷脂水解。有充分的文献证据，仅以数据驱动相关性的形式，表明界面的物理化学性质在水解速率中起主要作用。本项目的目的是为磷脂酶膜酶学中尚未解决的界面质量效应问题建立一个范例。基于最近对磷脂酶在胶束界面上活性的研究，我们假设了磷脂酶在胶束界面上的动力学方案和具体功能：三个关键的连续步骤是：1)酶-与囊泡结合形成E*，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，缔合速率常数为k2，解离速率常数为k-2；3)脂质水解，速率常数为k3。具体来说，E*L的缔合和解离是热激活过程，其能垒分别为5L和5R，因此k2 = k20 exp (-5L/kBTB), k-2 = k-20 exp (-5R/kBTB)，其中kBB为玻尔兹曼常数。膜结构决定了5L和5R的能量；因此速率常数k2和k-2；表面结合常数KS和k3（通过双层水化）。脂质类型和组成决定了膜的结构。因此，动力学参数KS、k2、k-2和k3与组分有关。因此，界面作用的机理细节源于E*L、E*和水化的膜结构依赖性质。目标是：1。采用不同比例的底物l -磷脂及其非水解d -对映体的混合物，开发一种新的磷脂酶动力学测定方法，以设计表面稀释系列。这种混合物解决了一个长期存在的问题，即在双层中改变界面衬底浓度的能力。测量活性与底物浓度，通过建立良好的pH-stat以及新的荧光测定，使用FRET（荧光共振能量转移）荧光团标记的磷脂。将提出的动力学方案所得到的模型与数据拟合，得到动力学参数。2. 确定k2和k-2对阿尼乌斯温度依赖性的影响。利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。检查微量量热数据与动力学数据的一致性。用电子自旋共振法测定双分子层水合作用，确定其与k3的关系及对k3的影响。这项工作的意义在于它有可能通过通过动力学参数表达界面物理化学性质的调节作用的新范式来阐明术语“界面质量效应”。这对人类健康很重要，因为水解产物具有多种生理功能，包括细胞信号传导、炎症、过敏、细胞凋亡和肿瘤发生。