MOLECULAR DYNAMICS OF LIPOLYTIC ENZYME CATALYSIS

动脉粥样硬化是美国的头号杀手，与动脉粥样硬化有关 体内许多脂类代谢途径的功能和 相应的脂解酶。尽管如此，人们对 脂解酶催化的分子动力学，这阻碍了 合理设计这些酶的基于机制的效应器 在动脉粥样硬化的研究和/或治疗中有价值。这个 在此提出的研究的主要目标是开发一种详细的 对界面脂蛋白脂肪酶分子动力学的认识 (LPL)和胆固醇酯酶(停止)催化，并确定如何 界面催化对生理激活剂和合成催化剂的响应 抑制剂。选择用于研究界面脂解动力学的系统 由混合胶束中含有的脂类对硝基苯酯组成， 提供以下实验优势：a)胶束处的水解性 用分光光度法可以连续跟踪表面，这使得 易于计算机化的反应监测和数据还原。b) 反应时间过程用综合形式描述 Michaelis-Menten方程，它允许计算界面 单次运行的参数Km和Vmax。将应用于 界面催化研究包括：a)酰基LPL的亲核捕获 和停酰中间体；b)溶剂同位素和pH速率对 确定LPL催化是否涉及质子转移机制；c) LPL和STOP催化剂的不可逆抑制；d)表征 直接测量Km和Vmax的过渡态模拟抑制 停止和LPL反应；e)确定LPL的效果 激活剂apoC-II和界面上的停止激活剂胆盐 米氏参数Km和Vmax；f)脂肪酸产物抑制 G)新型可逆和不可逆止动器的设计 属于胆固醇酯类似物的抑制剂；h)合成 硫代胆固醇酯作为仿生显色底物。 预计本申请中描述的实验将 介绍了一种研究界面LPL和STOP的新方法 反应动力学。拟议研究的长期目标是 利用对即将到来的界面上的酶催化的理解 从这些实验中设计出有效的基于机制的抑制剂 脂解酶可能是脂代谢研究中有价值的工具 或在动脉粥样硬化的药理学中。

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.