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; 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.

消化酶通过磷脂水解是一种界面现象，发生在脂质/胆汁盐处 人消化道中的骨料和溶剂界面。存在明确的证据，表明 界面特性（脂质表面浓度，尺寸，曲率，界面水合和电荷和脂质 构象）调节酶活性。但是，没有发展的功能关系，因为 迄今为止，酶学研究已在定义较差的底物骨料上进行。控制的因素 胶束界面处的磷脂酶动力学尚未确定。拟议的研究旨在 删除此缺点。物理化学表征和磷脂酶的互补研究 将对同一底物骨料系统进行动力学研究，从而弥合 两种类型的研究。这将影响目前将胶束视为胶束的生化测定方法 黑匣子。酶学中的一个基本问题是酶遵守哪些速率定律。没有假设或模型 可以在没有特征良好的底物聚集体的情况下进行测试。过去一年的磷脂酶C的结果 胆汁盐/脂质聚集体的酶动力学研究，其特征是时间分辨荧光淬灭 在物理化学骨料特性与酶活性之间显示出明显的定量相关性。 这些结果构成了当前建议的研究的基础，并激发了当前的研究，其具体目的是： （i）开发可靠的测定法以研究磷脂酶动力学并测试推定的预测 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物理化学特性与动力学之间 途径，因此显示界面如何调节酶活性。研究中包括其他 洗涤剂/磷脂模型底物系统，形成稳定的球状混合胶束，并定义明确 属性和几何形状，因此用作界面的控制，并导致最小歧义 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，溶液粘度和小角度的中子散射。确定的属性是 混合的胶束聚集数，胶束形状和尺寸，脂质的表面浓度，表面电荷， 界面水合，界面微度。这些属性在相当大的范围内的可调性 在开发界面时，将利用混合物组成和浓度 微结构 - 活动相关量表。长期目标是了解酶促的机制 界面处的催化。动力学途径的知识对于理解机制是至关重要的 因为提出的机制可以通过可靠的测定方法对动力学预测进行测试。建立 动力学途径在提供可用于提高效率的知识方面具有本质上的价值 代谢。改善和完善有关控制脂肪消化的知识对人类很重要 健康。