Mammalian phosphatidylinositol-specific phospholipases C (PIPLC) are key effectors of the action of growth factors, hormones and neurotransmitters. These enzymes are involved in transmission of extracellular signals into the cell interior by hydrolyzing phosphatidylinositol 4,5-bisphosphate, with formation of two intracellular signals, inositol 1,4,5-trisphosphate (IP3) and diacyl glycerol. Understanding the mechanism of action of these Ca2+-dependent PLC is crucial to understanding the regulation phenomena that modulate their activity during signaling events. The goal of this proposal is to obtain detailed insights into catalytic mechanism of the mammalian PLC by investigating their simpler analog from Streptomyces antibioticus. The model enzyme has been shown to have very similar product outcome, Ca-dependency and amino acid homology of key active site residues. Our approach will encompass a variety of methods including chemical syntheses of substrate analogs, site-directed mutagenesis, synthesis of structure-based inhibitors and determination of three-dimensional structure of the free protein and its complexes with cleavage-resistant substrate analogs. In particular, we will employ """"""""matched, substrate-enzyme mutagenesis"""""""" by studying effects of simultaneous modification of substrate and enzyme at the mutually interacting positions. We will also explain mechanistic basis for specificity of mammalian enzymes with regard to various phosphatidylinositol species, and the origin of the efficient sequential catalysis of two chemical steps involved in the overall process. Our results will enable comparisons with metal-ion independent PLC, and allow generalizations of chemical mechanisms among the species, with broader implications to enzymes that catalyze transfer of the phosphoryl group. In addition, we will develop a new fluorescence-based method for measurement of intracellular activities of PLC inside the living cell to allow direct time- and space-resolved observation of cellular responses to hormonal stimulation. Finally, we will synthesize strong inhibitors of PLC for applications as research tools in studies of cellular signaling, and as potential leads for future drug development against cancer. ? ?
