ACTIVATION OF PI-SPECIFIC PHOSPHOLIPASE C ENZYMES

磷脂酰肌醇特异性磷脂酶C催化磷酸肌醇裂解为二酰基甘油（蛋白激酶C的第二信使激活剂）和磷酸肌醇。该反应的机制是一个磷酸转移酶步骤，形成中间肌醇1,2-（环）磷酸（cIP），然后是一个环磷酸二酯酶反应，将cIP水解为磷酸肌醇（I-I- P）。哺乳动物酶作为pi信号的关键调节因子，在细胞生长和增殖中起着关键作用。由于PI-PLC酶是水溶性的，其正常底物磷酸肌苷位于膜中，因此它们的活性也可以通过脂质界面和其他膜定位信号成分来调节。我们感兴趣的是识别和表征界面激活这些酶的机制。为此，我们集中研究了环磷酸二酯酶反应，并开发了具有可溶性底物（甘油磷酸肌醇磷酸或短链PI）的磷酸转移酶测定系统，这些底物不会分裂成界面。监测非底物界面如何影响这些可溶性反应，可以将对酶的变构效应与界面底物的改变分离开来。待研究的主要PI-PLC酶包括来自苏云金芽孢杆菌的小的（35 kDa）细菌PI-PLC和最小的（85 kDa）哺乳动物同工酶PI-PLCdelta1。这种哺乳动物酶的活性位点模块在结构上与细菌酶非常相似，并且都表现出独特的动力学特征。多种物理技术（核磁共振、荧光、等温滴定量热法）将被用来表征(1)功能不同的磷脂激活剂和催化位点之间的空间关系；(2)脂质和溶剂激活剂诱导PI-PLC酶的构象变化；(3)活化剂对酶与双分子层体结合的影响；(4)为什么哺乳动物的酶会同时产生cIP和I-1-P；(5) G蛋白α亚基对plc - β 2和plc - β 3切割cIP的影响。这些研究的结果将为每个主要的PI-PLC类提供环磷酸二酯酶反应的完整图像，允许将涉及“可溶性”底物与磷酸肌肽裂解的这一步骤进行比较，并提供不同界面（包括“激活”磷脂以及其他蛋白质）如何改变这种化学反应的详细图像。这一信息对于找到在体内操纵这些酶的方法至关重要。

Phosphatidylinositol-specific phospholipase C enzymes catalyze the cleavage of phosphoinositides to diacylglycerol (a second messenger activator of protein kinase C) and inositol phosphates. The mechanism is sequential with a phosphotransferase step forming the intermediate inositol 1,2-(cyclic) phosphate (cIP) followed by a cyclic phosphodiesterase reaction to hydrolyze the cIP to inositol phosphate (I-I- P). The mammalian enzymes, as key regulators of PI-signaling have critical roles in cell growth and proliferation. Since PI-PLC enzymes are water-soluble and their normal substrates, phosphoinositides, are localized in membranes, their activity can also be modulated by lipid interfaces and other membrane localized signaling components. We are interested in identifying and characterizing the mechanisms by which interfaces activate these enzymes. To that end we have concentrated on the cyclic phosphodiesterase reaction and developed phosphotransferase assay systems with soluble substrates (glycerol phosphoinositol phosphates or short-chain PI) that do not partition into interfaces. Monitoring how non-substrate interfaces affect these soluble reactions allows separation of allosteric effects on the enzyme from alterations of the interfacial substrate. The primary PI-PLC enzymes to be studied include a small (35 kDa) bacterial PI-PLC from Bacillus thuringiensis and the smallest (85 kDa) of the mammalian isozymes, PI-PLCdelta1. The active site module of this mammalian enzyme is structurally quite similar to the bacterial enzyme, and both exhibit unique kinetic features. A variety of physical techniques (NMR, fluorescence, isothermal titration calorimetry) will be used to characterize (1) the spatial relationship between functionally distinct phospholipid activator and catalytic sites; (2) the conformational changes in PI-PLC enzymes induced by lipid and solvent activators; (3) the effect of activators on bulk binding of the enzymes to bilayers; (4) why mammalian enzymes generate cIP and I-1-P in parallel; and (5) the effect of G protein alpha subunit on PLC-beta2 and PLC-beta3 cleavage on cIP. The results of these studies will provide a complete picture of the cyclic phosphodiesterase reaction for each of the major PI-PLC classes, allow comparison of this step involving a 'soluble' substrate with phosphoinositide cleavage, and provide a detailed picture of how different interfaces, include 'activating' phospholipids as well as other proteins, alter this chemistry. This information will be critical in finding ways to manipulate these enzymes in vivo.