Interface Quality Effects in Phospholipase Membrane Enzymology

说明(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酶。有充分的文献证据，仅以数据驱动的关联的形式，界面物理化学性质在水解率中起主要作用。本项目的目标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。根据最近完成的关于胶束界面磷脂酶活性的工作，提出了双分子膜界面的动力学方案和特殊功能假设如下：1)酶与囊泡结合形成E*，平衡结合常数KS；2)E*与活性部位的脂类结合，形成界面络合物E*L，结合速率常数分别为K2和k-2；以及3)脂水解速率常数K3。具体地说，E*L的缔合和解离分别是具有5L和5R能垒的热激活过程，使得k2=k20exp(-5L/kBTB)和k-2=k-20exp(-5R/kBTb)，其中kbb是玻耳兹曼常数。膜结构定义了能量5L和5R；从而定义了速率常数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了膜的结构。因此，动力学参数KS、K2、K-2和K3与组成有关。因此，界面作用的机理细节来源于膜结构依赖于E*L、E*和水化的性质。目的是：1.建立一种新的磷脂酶动力学分析方法，利用底物L-磷脂及其非水解性D-对映体按不同比例混合设计表面稀释系列。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。通过成熟的pH恒定以及使用荧光共振能量转移(FRET)荧光团标记的磷脂的新的荧光分析，测量活性与底物浓度的关系。将所提出的动力学方案得到的模型与数据进行拟合，得到动力学参数。2.确定K_2和K_2的阿累尼乌斯温度依赖性的影响。用新型微量热法独立表征了配合物E*L，得到了E*L的生成自由能，检验了微量热法数据与动力学数据的一致性。用电子自旋共振测量双分子层水合作用，以确定与K3的相关性和对K3的影响。这项工作的意义在于，它有可能通过界面物理化学性质的调节作用通过动力学参数来表示的新范式来解释术语“界面质量效应”。这对人类健康很重要，因为水解物具有多种生理功能，包括细胞信号、炎症、过敏、细胞凋亡和肿瘤发生。

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.