STRUCTURE-FUNCTION OF ADP-RIBOSYL CYCLASE AND HOMOLOGS

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ADP-ribosyl cyclase belongs to a family of homologous proteins important in mediating Ca2+ signaling in cells. The other members of the family include the differentiation antigens CD38 (on lymphocytes) and BST1/BP3 (on bone marrow cells). In addition to structural homology all members of the family share functional similarities as well. They all are enzymes catalyzing the synthesis of cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), two structurally and functionally distinct Ca2+ messengers responsible for mobilizing different types of intracellular Ca2+ stores. The cADPR- and NAADP- sensitive Ca2+ stores have now been shown to be ubiquitously present in more than 40 different cell types from protozoart to human cells, indicating their general relevance. The cyclase family of enzymes is also catalytically novel. Contrary to normal enzymes which generally catalyze the conversion of one specific substrate to one product, the members of the family can use more than one substrate and convert them into structurally and functionally distinct products. Thus, the cyclase not only can cyclize NAD into cADPR, it can also use NADP as substrate and catalyze a base- exchange reaction with nicotinic acid, producing NAADP. Which catalytic path the cyclase takes is determined by pH. We have recently solved the crystal structure of the Aplysia cyclase and are in a position to characterize structure-function relationships of this novel multifunctional enzyme. Detailed knowledge of its catalytic mechanism is likely to have important ramifications in our understanding of Ca2 signaling mechanisms in cells. Specific aims are: l. To characterize the active sites of the Aplysia cyclase and human CD38. 2. To alter the active site by mutagenesis and assess the resulting changes in catalysis. 3. To determine the structure of the active site of the cyclase by X-ray crystallography. 4. To develop an inducible expression system of the cyclase for use in physiological studies.