INTERFACIAL ACTIVATION OF WATER SOLUBLE PHOSPHOLIPASES

细菌磷脂酶是参与细菌毒力和有机磷循环的脂溶酶。它们通常与哺乳动物参与信号转导的磷脂酶具有相似的动力学行为和有限的序列和结构同源性。这些酶的一个重要方面是，虽然它们通常是水溶性的，但它们在界面上进行催化。在许多情况下，酶与界面在不同位点的相互作用先于底物与催化位点的结合。本研究旨在从分子水平上了解蜡样芽孢杆菌非特异性磷脂酶C （PLC）和色褐链霉菌磷脂酶D （PLD）这两种水溶性脂溶酶的复杂界面行为。核磁共振、荧光、CD和定点诱变技术将与动力学分析结合使用，研究磷脂底物、活化剂和抑制剂与每种酶的相互作用。需要解决的一般问题包括(i)磷脂变构界面位点的分子性质是什么，变构界面位点是如何发生的，以及变构信号是如何传递到活性位点的？（Ii）给定活性位点的结构，是否可以系统地改变底物特异性或化学反应性？（iii）从磷脂激活剂和酶活性位点的角度来看，磷脂与这些酶结合的关键参数是什么？这些研究结果将为PLC和PLD酶的界面行为和催化机制提供见解。将开发界面结合位点的分子图谱，以及对磷脂酶活性后产物（包括脂质第二信使）和底物物理特性的扎实理解。

Bacterial phospholipases are lipolytic enzymes involved in bacterial virulence as well as organophosphate recycling. They often share similar kinetic behavior and limited sequence and structural homology with mammalian phospholipases that are involved in signal transduction. An important aspect of these enzymes is that while they are in general water- soluble, they carry out their catalysis at an interface. In many cases interaction of the enzyme with the interface at a distinct site precedes substrate binding to the catalytic site. The proposed work seeks to understand on a molecular level the complex interfacial behavior of two water-soluble lipolytic enzymes: Bacillus cereus non-specific phospholipase C (PLC) and Streptomyces chromofuscus phospholipase D (PLD). NMR, fluorescence, CD, and site-directed mutagenesis techniques will be used in conjunction with kinetic analyses to investigate phospholipid substrate, activator, and inhibitor interactions with each enzyme. General questions to be addressed include (i) what is the molecular nature of phospholipid allosteric interfacial site and how is the allosteric interfacial site and how is the allosteric signal transferred to the active site? (Ii) given the architecture of the active site, can substrate specificity or chemical reactivity be altered in a systematic way? And (iii) what are the critical parameters for phospholipid binding to these enzymes, both from the point of view of phospholipid activator and enzyme active sites? The results of these studies should provide insights into the interfacial behavior and catalytic mechanisms for the PLC and PLD enzymes. A molecular picture will be developed for interfacial binding sites as well as a solid understanding of how products (that include lipid second messengers), and substrate physical characteristics after phospholipase activity.