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.
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