Interface Quality Effects in Phospholipase Membrane Enzymology

磷脂酶膜酶学中的界面质量效应

• 批准号: 8076584
• 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/8076584
• 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. 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.

描述（由申请人提供）：磷脂酶是消化酶以及外周膜酶，其在膜-水界面处催化磷脂水解。有充分的证据表明，仅以数据驱动的相关性的形式，界面物理化学性质在水解速率中起着主要作用。该项目的目标是为磷脂酶膜酶学中尚未解决的界面质量影响问题建立一个范例。基于最近完成的关于胶束界面处磷脂酶活性的工作，双层界面的动力学方案和特定功能假设如下：三个关键的连续步骤是：1）酶与囊泡结合形成具有平衡结合常数KS的E*； 2) E*在活性位点结合脂质形成界面复合物E*L，其缔合和解离速率常数分别为k2和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，能垒分别为 5L 和 5R，因此 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5L和5R；以及速率常数 k2 和 k-2；表面结合常数 KS 和 k3（通过双层水合）。脂质类型和成分决定了膜结构。因此，动力学参数 KS、k2、k-2 和 k3 取决于成分。因此，界面作用的机制细节源于 E*L、E* 和水合作用的膜结构依赖性特性。目标是： 1. 使用不同比例的底物 L-磷脂及其非水解 D-对映异构体的混合物来设计表面稀释系列，开发一种新颖的磷脂酶动力学测定方法。这种混合物解决了长期存在的能够改变双层中界面底物浓度的问题。通过完善的 pH 统计器以及采用 FRET（荧光共振能量转移）荧光团标记的磷脂的新型荧光测定法，测量活性与底物浓度的关系。将所提出的动力学方案产生的模型与数据进行拟合并获得动力学参数。 2. 确定 k2 和 k-2 的 Arhenius 温度依赖性的影响。通过新型微量热法独立表征复合物 E*L 并获得 E*L 形成的自由能。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 的相关性和影响。这项工作的意义在于它有可能通过新的范式来阐明“界面质量效应”这一术语，即界面物理化学性质的调节作用是通过动力学参数来表达的。这对人类健康非常重要，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。 公共健康相关性：脂解活性涉及一系列正常生理和病理生理细胞功能，包括细胞信号传导、炎症、过敏、细胞凋亡和肿瘤发生。阐明膜理化特性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。