MOLECULAR DYNAMICS OF LIPOLYTIC ENZYME CATALYSIS

动脉粥样硬化是美国的头号杀手， 身体的脂质代谢途径的主体的功能， 相应的脂肪分解酶。 尽管如此，人们对它知之甚少。 脂肪分解酶催化的分子动力学，这阻碍了 这些酶的基于机制的效应物的合理设计， 在动脉粥样硬化的研究和/或治疗中具有价值。 的 本文提出的研究的主要目标是制定一个详细的 了解界面脂蛋白脂酶的分子动力学 (LpL)和胆固醇酯酶（CEase）催化，并确定如何 界面催化响应生理活化剂和合成 抑制剂的 选择用于研究界面脂肪分解动力学的系统 由包含在混合胶束中的脂质对硝基苯基酯组成， 提供以下实验优点：a）在胶束处水解 表面可以通过分光光度法连续跟踪，这使得 反应监测和数据简化易于计算机化。 B） 反应时间过程用积分形式描述， Michaelis-Menten方程，该方程允许计算界面 参数Km和Vmax。 将被应用于 研究界面催化包括：a）酰基-LpL亲核捕获 和酰基-CE酶中间体; B）溶剂同位素和pH速率效应， 确定LpL催化是否涉及质子转移机制; c） LpL和CEase催化的不可逆抑制; 通过直接测量Km和Vmax的过渡态类似物抑制 CEase和LpL反应; 活化剂apoC-II和CEase活化剂胆汁盐 米氏参数Km和Vmax; f）脂肪酸产物抑制 g）新型可逆和不可逆CEase的设计 作为胆固醇酯类似物的抑制剂; 硫代胆固醇酯作为仿生显色CEase底物。 预期本申请中描述的实验将 介绍了一种研究界面LpL和CEase强有力新方法 反应动力学 拟议研究的长期目标是 利用对酶催化作用的理解， 从这些实验中设计出有效的基于机制的抑制剂， 脂肪分解酶可能是脂质代谢研究中有价值的工具 或动脉粥样硬化的药理学。

Atherosclerosis is the #1 killer in the US, and involves the aberrant functioning of a host of the body's lipid metabolic pathways and 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 by 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 Km and Vmax 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 Km and Vmax 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 Km and Vmax; f) Fatty acid product inhibition of LpL at interfaces; g) Design of novel reversible and irreversible CEase inhibitors that are cholesteryl ester analogs; h) Synthesis of thiocholesteryl esters as biomimetic chromogenic CEase substrates. 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.