Interface Quality Effects in Phospholipase Membrane Enzymology

磷脂酶膜酶学中的界面质量效应

• 批准号: 8461141
• 负责人: Radha Ranganathan
• 金额: $ 10.49万
• 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/8461141
• 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）：磷脂酶是消化的以及外周膜酶，可在膜 - 水界面催化磷脂水解。只有数据驱动相关性的形式有充分的文档证据，即界面理化特性在水解速率中起着重要作用。该项目的目的是为磷脂酶膜酶学中界面质量效应尚未解决的问题建立一个范式。基于最近在胶束界面上磷脂酶活性的结论，将双层的动力学方案和界面的特定功能假设如下：三个关键的顺序步骤是：1）酶与囊泡结合以E*形成E*，均与平衡结合常数ks形成E*； 2）E*在活性位点结合脂质，分别与缔合和解离速率常数K2和K-2形成界面复合物E* L； 3）脂质水解速率常数K3。具体而言，E*L的关联和分离分别与5L和5R能量屏障的热激活过程，因此K2 = K20 EXP（-5L/KBTB）和K-2 = K-2 = K-20 EXP（-5R/KBTB），其中KBB是Boltzmann常数。膜结构定义了能量5L和5R；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）。脂质类型和组成定义膜结构。因此，动力学参数KS，K2，K-2和K3与组成有关。因此，该界面作用的机械细节源自E*L，E*和水合的膜结构依赖性特性。目的是：1。使用底物L-磷脂的混合物及其非水解d-Enantiomers的磷脂酶动力学开发一种新颖的测定法，以各种比例设计表面稀释系列。这种混合物是解决长期存在的问题的解决方案，即可以改变双层中的界面底物浓度。测量活性与底物浓度，通过良好的pH-stat以及采用用FRET标记的磷脂（荧光共振能传递）荧光团标记的磷脂的新荧光测定。拟合来自提出的动力学方案产生的模型，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。通过新型的微钙化来独立表征复合物E*L，并获得E*L形成的自由能。检查微钙化数据与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。这项工作的重要性是通过新范式阐明术语“界面质量效应”的潜力，即接口物理化学特性的调节作用通过动力学参数表达。这对人类健康非常重要，因为水解产物执行了几种生理功能，包括细胞信号，炎症，过敏，凋亡和肿瘤发生。