STRUCTURE-FUNCTION OF ADP-RIBOSYL CYCLASE AND HOMOLOGS

ADP-核糖环化酶是细胞内重要的钙信号转导同源蛋白家族。该家族的其他成员包括分化抗原CD38(在淋巴细胞上)和BST1/BP3(在骨髓细胞上)。除了结构上的同源性，该家族的所有成员在功能上也有相似之处。它们都是催化合成环状ADP-核糖(CADPR)和烟酸腺嘌呤二核苷酸磷酸(NAADP)的酶，这两种结构和功能不同的钙信使负责动员不同类型的细胞内钙库。CADPR和NAADP敏感的钙离子储存库现在已经被证明普遍存在于从原生动物到人类细胞的40多种不同类型的细胞中，这表明它们普遍相关。环酶家族在催化方面也是新的。与通常催化一种特定底物转化为一种产品的正常酶相反，该家族的成员可以使用多种底物，并将它们转化为结构和功能不同的产品。因此，该环化酶不仅可以将NAD环化成cADPR，而且还可以以NADP为底物，催化与烟酸的碱基交换反应，生成NAADP。环化酶的催化路径由pH决定。我们最近已经解决了海藻环化酶的晶体结构，并能够表征这种新型多功能酶的结构-功能关系。对其催化机制的详细了解可能会对我们理解细胞内的钙信号机制产生重要的影响。具体目标是：L。研究海藻环化酶和人CD38的活性部位。2.通过诱变改变活性部位，并评估由此产生的催化变化。3.用X射线结晶学方法确定环化酶活性中心的结构。4.建立用于生理学研究的环化酶诱导表达系统。

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.