The inositol-related pathways are extremely complicated and important biologically. The key step in these pathways is the conversion of phosphatidylinositol (PI) to diacylglycerol, inositol 1,2-cyclic phosphate (IcP), and inositol 1-phosphate (IP), catalyzed by PI-specific phospholipase C (PI-PLC). The goal for the next period is to extend our stereochemical studies of PI-PLC to structure-function analyses of three enzymes which catalyze three variations of this process: bacterial PI-PLC from B. thuringiensis, mammalian PI-PLC-beta1 from bovine brain, and annexion III from human lung. Annexion III is a Ca2+-dependent phospholipid binding protein in the lipocortin/annexion family but has been demonstrated to catalyze reactions similarly to PI-PLC except that the substrate is glycerophosphoinositol (GroPI) instead of Pl. The long range goals are to understand how these enzymes catalyze their reactions, and what aims for the next granting period: (1) To overexpress annexion II in E. coli. The B. thuringiensis PI-PLC will be purified from an overproducing E. coli strain given to us by Dr. Henner at Genentech. (2) To develop continuous assay methods, and to establish the basic kinetic properties of the three enzymes. (3) To synthesize three types of substrate analogs, with modifications on the inositol moiety: deoxy analogs, configurational isomers, and epoxy analogs. (4) To probe the substrate specificity of all three enzymes using the deoxy analogs of substrates. (5) To probe the stereospecificity of all three enzymes using the configuration a isomers of substrates. The steric courses of the reactions catalyzed by annexion III will also be elucidated by employing 170 and 180 isotopes. (6) To test the epoxy analogs of PI and GroPI as irreversible inhibitors. The potent inhibitors will then be synthesized in radioactive forms and used to label the enzyme. The labelled enzymes will be digested and the site of modification identified. (7) To initiate site-directed mutagenesis studies with the bacterial Pi-PLC and annexion III. The first target residues will be chosen based on sequence homology, the results of chemical modification, and crystal structures from other laboratories. (8) To attempt proton NMR analyses, and to try growing crystals, of the free enzymes and enzyme-inhibitor complexes, for bacterial PI-PLC and annexion III (both with molecular weight ca. 35,000).
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