Phospholipase Kinetics in Mixed Micelles

摘要磷脂被消化酶降解是发生在脂质/胆盐之间的界面现象。 人体消化道中的聚集体和溶剂界面。确凿的证据表明， 界面性质(脂类表面浓度、大小、曲率、界面水化、电荷和脂类 构象)调节酶活性。然而，没有开发出函数关系，因为 到目前为止，酶学研究一直是在定义不清的底物聚集体上进行的。控制因素 胶束界面上的磷脂酶动力学尚未建立。拟议的研究旨在 消除这一缺点。理化性质与磷脂酶的互补性研究 将对相同底物聚合体系进行动力学研究，从而弥合 两种类型的研究。这将影响目前将胶束作为一种 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 可以在没有表征良好的底物聚集体的情况下进行测试。过去一年磷脂酶C的研究结果 时间分辨荧光猝灭法研究胆盐/脂聚集体的酶动力学 结果表明，酶的物理化学聚集性质与酶活性之间存在明显的定量关系。 这些结果构成了本拟议研究的基础和动力，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，并检验假定的 Michaelis-Menten模型在界面酶动力学中的应用(II)确定定量关联式 磷脂酶活性与胶束底物理化性质的关系及其动力学 从而展示了界面如何调节酶的活性。研究中包括的还有其他 洗涤剂/磷脂模型底物系统，形成稳定的球状混合胶束，定义良好 属性和几何图形，从而作为界面的控制并导致最小的歧义 在对结果的解释中。磷脂酶动力学将用pH-Stat法测定。胶束 通过一组互补的技术来执行表征：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。确定的属性包括 混合胶束聚集数、胶束形状和大小、脂类表面浓度、表面电荷、 界面水化、界面微粘度。这些属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 微结构-活性相关量表。长期的目标是了解酶的作用机制。 界面上的催化作用。对动力学途径的了解是理解机制的基础， 因为所提出的机制可以做出动力学预测，而这些预测可以用可靠的分析来检验。建立 动力学路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。改进和完善控制脂肪消化的知识对人类很重要 健康。

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.