大分子拥挤环境中脂肪酸酐自催化水解异常链长依赖效应的热力学机制

An unusual chain-length-dependent autocatalytic hydrolysis of fatty acid anhydrides induced by polyethylene glycol was found by our group recently. Therefore, further disclosing the thermodynamic mechanism behind the autocatalytic hydrolysis of fatty acid anhydrides in macromolecular crowding becomes an important scientific problem. In this proposal, the research starts with preparation of different kinds of macromolecular crowded solutions with varied structure and concentration. Systematical research will focus on the physiochemical properties of crowded media as well as the energy both in vesicles formation and in the hydrolysis of fatty acid anhydrides catalyzed by vesicles in the presence of macromolecular crowding. The driving force involved in the change of the enthalpy, the entropy and the Gibbs energy will be analyzed to find how the macromolecular crowding agents influence and control the autocatalytic hydrolysis of fatty acid anhydrides, which results in the unusual chain-length-dependent effect. Current research mainly focuses on the effect of macromolecular crowding agents on the structural change of proteins or nuclear acid as well as the kinetics of enzyme-catalyzed reactions. On the other hand, autocatalytic hydrolysis of fatty acid anhydrides catalyzed by the spontaneously formed vesicles in macromolecular crowded solutions remains unknown. Therefore, it is of great importance to discover the thermodynamic basis for this unusual chain-length-dependent autocatalytic hydrolysis of fatty acid anhydrides in macromolecular crowded conditions. Meantime, this research will open up an area to study how macromolecules control the reaction and self-assembly of small molecules. Moreover, it will provide useful information for understanding the hydrolysis of fats in small intestine where it is crowded and occupied by many macromolecular species.

申请人前期发现聚乙二醇构建的拥挤环境诱导脂肪酸酐自催化水解出现了异常链长依赖现象，因此进一步探索其背后的热力学机制是一个很有意义的科学问题。本项目从构建不同类型大分子拥挤环境入手，系统研究拥挤环境内在物理性质变化、脂肪酸囊泡组装及二者协同诱导脂肪酸酐水解动力学过程中的焓、熵、自由能变化及交互影响的驱动力差异，试图揭示大分子拥挤诱导不同结构脂肪酸酐自催化水解反应的热力学本源。长期以来，大分子拥挤效应研究集中在其对蛋白质、核酸结构变化及酶催化反应的影响，尚未关注其控制小分子自组装及化学反应；而国际上脂肪酸酐水解及脂肪酸囊泡组装的研究均未考虑大分子拥挤效应。本研究的意义在于揭示系列大分子拥挤环境对不同结构脂肪酸酐自催化水解反应中调控规律的物理化学机制；同时也将大分子拥挤效应研究领域拓展向大分子调控小分子自催化反应及自组装研究；或许也对于理解人体肠道内大分子存在下脂肪的消化吸收有着重要的科学意义。

申请人前期发现了一种大分子拥挤环境中脂肪酸酐水解的异常链长依赖现象(2014年)，因此探索其背后的热力学机制是个有意义的科学问题。本项目的研究意义在于，围绕大分子拥挤导致的脂肪酸酐水解异常链长依赖现象这个核心，从一个集“大分子拥挤”、“囊泡自组装”、“囊泡界面水解反应”为一体的不同体系出发，通过系统研究大分子拥挤环境中各种结构脂肪酸参与的自催化水解、酶催化反应及囊泡自组装等，探讨其中的热力学参数变化及交互影响的驱动力差异，试图揭示大分子拥挤介质中界面催化产生脂肪酸的异常链长依赖效应的普遍性及内在热力学机制。 .按照项目计划书，主要从聚乙二醇拥挤环境中脂肪酸酐自催化水解反应、多糖拥挤环境中酸酐自催化水解反应、大分子拥挤环境中脂肪酸与蛋白质相互作用、大分子拥挤环境中脂肪酶催化甘油酯水解反应、大分子拥挤环境中维生素 E 醋酸酯的酯解反应及体外消化等五个体系入手，基于ITC等热力学方法进行了系统的研究。结果表明，在大分子拥挤介质参与的反应体系中，脂肪酸异常链长依赖效应具有普遍性，但不同体系各具特点。主要的结论有：（1）在脂肪酸囊泡自催化酸酐水解反应中，拥挤度增加使中链脂肪酸生成速率减慢，而长链脂肪酸生成速率加快。（2）大分子拥挤介质提高了中链脂肪酸与血清蛋白的结合常数，却降低了长链不饱和脂肪酸的结合常数，显示出大分子拥挤介质对蛋白质结构及其结合脂肪酸热力学状态的控制。（3）某些多糖使脂肪酶催化反应速率及催化效率（kcat/Km值）较稀溶液中增加，同时也提高了脂肪酸的生物可及率。.总之，大分子拥挤介质既能调控囊泡界面的酸酐水解反应，也能调控界面酶催化甘油三酯水解反应。项目较好地完成了预定研究目标(已发表SCI论文6篇，参加学术交流国外4次、国内5次)。本研究不仅加深了对传统大分子拥挤效应的系统理解，且启发人们利用大分子拥挤效应调控界面酶的特性而提高反应速率及效率。这对于开拓新的前沿研究与应用方向，有着极其重要的科学意义。