Phospholipase Kinetics in Mixed Micelles

混合胶束中的磷脂酶动力学

• 批准号: 7131845
• 负责人: Radha Ranganathan
• 金额: $ 9.77万
• 依托单位: CALIFORNIA STATE UNIVERSITY NORTHRIDGE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7131845
• 关键词: chemical kinetics; enzyme activity; enzyme substrate complex; lipolysis; mathematical model; micelles; mitotic spindle apparatus; phospholipase A2; phospholipase C; physical chemical interaction; time resolved data

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.

消化酶对磷脂的水解是发生在脂质/胆盐界面的一种界面现象 聚集体和溶剂界面在人体消化道。确凿的证据表明， 界面性质（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，没有建立功能关系，因为 迄今为止，酶学研究是在定义不明确的底物聚集体上进行的。控制因素 磷脂酶在胶束界面的动力学尚待建立。拟议的研究旨在 消除这个缺点。磷脂酶和理化特性的补充研究 将对同一基质聚集体系统进行动力学研究，从而弥合 两种研究。这将影响目前将胶束作为生物活性物质处理的生物化学测定方法。 黑匣子酶学中的一个基本问题是酶遵循什么速率定律。无假设或模型 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，其特征在于时间分辨荧光猝灭 显示了明确的物理化学聚集体性质和酶活性之间的定量相关性。 这些结果构成了目前拟议研究的基础和动机，其具体目标是： (i)开发可靠的磷脂酶动力学研究方法，并对假定的 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之间的关系以及动力学 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，其形成稳定的球形混合胶束， 属性和几何形状，从而作为界面的控制，并导致最小的歧义 对结果的解释。将通过pH-Stat方法测量磷脂酶动力学。胶束 通过一组互补的技术进行表征：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。所确定的性质是 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，界面微粘度。这些特性在相当大的范围内的可调性 根据混合物的组成和浓度，将在开发界面时加以利用 微观结构-活性相关性量表长期的目标是了解酶的机制， 界面催化作用动力学途径的知识是理解机制的基础， 因为所提出的机制可以进行动力学预测，这些预测可以用可靠的测定法进行测试。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 新陈代谢的过程。提高和完善什么控制脂肪消化的知识对人类来说是重要的 健康