Phospholipase Kinetics in Mixed Micelles

混合胶束中的磷脂酶动力学

• 批准号: 7455724
• 负责人: Radha Ranganathan
• 金额: $ 15.15万
• 依托单位: CALIFORNIA STATE UNIVERSITY NORTHRIDGE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7455724
• 关键词: Binding Sites; Biochemical; Biological Assay; Boxing; Catalysis; Charge; Chemicals; Complex; Detergents; Digestion; Dimyristoylphosphatidylcholine; Electron Spin Resonance Spectroscopy; Enzymatic Biochemistry; Enzyme Kinetics; Enzymes; Family suidae; Fatty acid glycerol esters; Fluorescence; Foundations; Gastrointestinal tract structure; Goals; Health; Human; Hydration status; Hydrolysis; Investigation; Kinetics; Knowledge; Laws; Lipid Binding; Lipids; Literature; Measures; Metabolic; Metabolism; Methods; Micelles; Modeling; Molecular Conformation; Neutrons; Numbers; Pancreas; Pathway interactions; Phospholipase; Phospholipase A2; Phospholipase C; Phospholipids; Property; Purpose; Range; Rate; Relative (related person); Reporting; Research Design; Shapes; Sodium; Sodium Cholate; Sodium Deoxycholate; Solutions; Solvents; Specificity; Surface; System; Taurocholate Sodium; Techniques; Testing; Time; Ursidae Family; Viscosity; bile salts; chemical binding; enzyme activity; improved; size; time use

Phospholipid hydrolysis by digestive enzymes is an interface phenomenon that occurs at the lipid/bile-salt aggregate and solvent interface in the human digestive tract. Unequivocal evidence exists, showing that interface properties (lipid surface concentration, size, curvature, interface hydration and charge and lipid conformation) modulate enzyme activity. However there are no functional relations developed because enzymology studies to date have been performed on poorly defined substrate aggregates. Factors that control phospholipase kinetics at micellar interfaces are yet to be established. The proposed studies are designed to remove this shortcoming. Complementary studies of physico-chemical characterization and phospholipase kinetics studies on the same substrate aggregate systems will be conducted, thus bridging the gap between the two types of studies. This will impact the approach to biochemical assays that presently treat micelles as a black box. A basic question in enzymology is what rate law is obeyed by an enzyme. No hypothesis or model can be tested without well-characterized substrate aggregates. Results of the past year on phospholipase C enzyme kinetic studies on bile salt/lipid aggregates, characterized by time-resolved fluorescence quenching showed a clear quantitative correlation between the physicochemical aggregate properties and enzyme activity. These results form the foundation of and motivate the present proposed studies, the specific aims of which are: (i) Develop reliable assays for investigating phospholipase kinetics and test the predictions of the putative Michaelis-Menten model applied to interface enzyme kinetics, (ii) Determine the quantitative correlation between phospholipase activity and the physicochemical properties of the micellar substrate and the kinetic pathway and thus show how the interface modulates enzyme activity. Included in the studies are other detergent/phospholipid model substrate systems, that form stable, globular mixed micelles with well-defined properties and geometries, serving thereby as controls of the interface and leading to a minimum of ambiguity in the interpretation of results. Phospholipase kinetics will be measured by the pH-Stat method. Micelle characterization is performed by a complementary set of techniques: time-resolved fluorescence quenching, electron spin resonance, solution viscosity, and small-angle neutron scattering. The properties determined are the mixed micelle aggregation numbers, micelle shape and size, surface concentration of lipids, surface charge, interface hydration, interface microviscosity. The tunability of these properties over a considerable range depending on mixture compositions and concentrations will be exploited in developing the interface microstructure-activity correlation scale. The long-term goal is to understand the mechanism of enzymatic catalysis at interfaces. Knowledge of the kinetic pathway is fundamental to understanding the mechanism, because proposed mechanisms make kinetic predictions which can be tested with reliable assays. Establishing the kinetic pathway is intrinsically valuable in providing knowledge that can be used in improving the efficiency of metabolism. Improving and refining the knowledge of what controls digestion of fats is important to human health.

消化酶水解磷脂是脂质/胆盐处发生的界面现象 人体消化道中的聚集体和溶剂界面。存在明确的证据表明 界面特性（脂质表面浓度、尺寸、曲率、界面水合和电荷以及脂质 构象）调节酶活性。然而，没有开发出功能关系，因为 迄今为止，酶学研究是在不明确的底物聚集体上进行的。控制因素 胶束界面处的磷脂酶动力学尚未确定。拟议的研究旨在 去掉这个缺点。理化特性和磷脂酶的补充研究 将在相同的基质聚集体系统上进行动力学研究，从而弥合两者之间的差距 两种类型的研究。这将影响目前将胶束视为一种生物化学检测方法。 黑匣子。酶学中的一个基本问题是酶遵循什么速率定律。没有假设或模型 可以在没有充分表征的底物聚集体的情况下进行测试。去年磷脂酶 C 的结果 胆汁盐/脂质聚集体的酶动力学研究，以时间分辨荧光猝灭为特征 显示出理化聚集体特性和酶活性之间存在明显的定量相关性。 这些结果构成了当前拟议研究的基础并激励了这些研究，其具体目标是： (i) 开发可靠的分析方法来研究磷脂酶动力学并测试假定的预测 Michaelis-Menten 模型应用于界面酶动力学，(ii) 确定定量相关性 磷脂酶活性与胶束底物的理化性质以及动力学之间的关系 途径，从而显示界面如何调节酶活性。研究中还包括其他 洗涤剂/磷脂模型底物系统，形成稳定的球状混合胶束，具有明确的边界 属性和几何形状，从而作为界面的控制并导致最小的歧义 在结果的解释中。磷脂酶动力学将通过 pH-Stat 方法测量。胶束 表征是通过一组互补的技术进行的：时间分辨荧光猝灭， 电子自旋共振、溶液粘度和小角中子散射。确定的属性是 混合胶束聚集数、胶束形状和大小、脂质表面浓度、表面电荷、 界面水化、界面微观粘度。这些属性在相当大的范围内可调 取决于混合物的成分和浓度，将用于开发界面 微观结构-活性相关量表。长期目标是了解酶促作用的机制 界面催化。了解动力学途径是理解机制的基础， 因为所提出的机制可以做出动力学预测，可以通过可靠的分析进行测试。建立 动力学途径在提供可用于提高效率的知识方面具有内在价值 的新陈代谢。提高和完善控制脂肪消化的知识对人类很重要 健康。