Atherosclerosis is the #1 killer in the US, and involves the aberrant functioning of a host of the body's lipid metabolic pathways an corresponding lipolytic enzymes. Despite this, little is known of the molecular dynamics of lipolytic enzyme catalysis, which hampers the?rational design of mechanism-based effectors of these enzymes that may be of value in the investigation and/or therapy of atherosclerosis. The primary objective of the research proposed herein is to develop a detailed understanding of the molecular dynamics of interfacial lipoprotein lipase (LpL) and cholesterol esterase (CEase) catalysis, and to determine how interfacial catalysis responds to physiological activators and synthetic inhibitors. The system chosen to study interfacial lipolytic kinetics consists of lipid p-nitrophenyl esters contained in mixed micelles, which offer the following experimental advantages: a) Hydrolysis at the micelle surface can be followed continuously be spectrophotometry, which makes reaction monitoring and data reduction amenable to computerization. b) Reaction timecourses are described by the integrated form of the Michaelis-Menten equation, which allows calculation of the interfacial parameters K*m and V*max from single runs. Probes that will be applied to study interfacial catalysis include: a) Nucleophilic trapping of acyl-LpL and acyl-CEase intermediates; b) Solvent isotope and pH-rate effects to determine whether LpL catalysis involves a proton transfer mechanism; c) Irreversible inhibition of LpL and CEase catalyses; d) Characterization of transition state analog inhibition by direct measurement of K*m and V*max for CEase and LpL reactions; e) Determination of the effect of the LpL activator apoC-II and the CEase activator bile salts on the interfacial Michaelis-Menten parameters K*m and V*max; f) Fatty acid product inhibition of LpL at interfaces; g) Design of novel reversible and irreversible CEase inhibitors that are cholestery ester analogs. h) Synthesis of thiocholesteryl esters are biomimetic chromogenic CEase substrateds. It is anticipated that the experiments described in this application will introduce a powerful new method for investigating interfacial LpL and CEase reaction dynamics. The long term goal of the proposed research is to utilize the understanding of enzyme catalysis at interfaces that will come from these experiments to design potent mechanism-based inhibitors of lipolytic enzymes that may be valuable tools in lipid metabolism research or in the pharmacology of atherosclerosis.
