Phospholipase Kinetics in Mixed Micelles

California State University Northridge, Northridge CA

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Abstract

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.