YEAST TWO-HYBRID ANALYSIS OF ATP SULFURYLASE

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。ATP硫酰化酶催化硫酸盐活化的第一步，将ATP的腺苷基部分转移到硫酸盐上，形成5 -磷酸硫酸腺苷（APS）和焦磷酸盐。随后，APS激酶介导γ的转移；磷酸基的ATP到APS形成3¿¿磷酸腺苷5'磷酸硫酸酯（PAPS）和ADP。ATP硫化酶反应是非常不利的，Keq估计为1 × 10-8，因此不同的机制已经进化到促进APS的形成。在大肠杆菌中，APS的形成与GTP的水解相结合。在高等真核生物中，ATP硫化酶和APS激酶融合在一个多肽上，通过将APS从ATP硫化酶活性位点引导到APS激酶活性位点来促进APS的合成。我们从酿酒酵母中解出ATP硫化酶的结构。这种酶采用同六聚体排列。每个单体的c端结构域虽然没有催化活性，但在结构上与APS激酶非常相似，并且发现了一个表面凹槽，可以作为ATP硫化酶和APS激酶样结构域之间的底物通道。虽然这种酶在进化上似乎与一种双功能酶有关，但由于APS激酶结构域催化失活，因此如何促进APS的形成尚不清楚。利用酵母双杂交分析，我们计划研究酵母ATP硫酰化酶是否与其他蛋白质相互作用以促进APS合成反应。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ATP sulfurylase catalyzes the first step in the activation of sulfate by transferring the adenylyl moiety of ATP to sulfate to form adenosine 5¿¿phosphosulfate (APS) and pyrophosphate. Subsequently, APS kinase mediates transfer of the gamma;phosphoryl group of ATP to APS to form 3¿¿ phosphoadenosine 5' phosphosulfate (PAPS) and ADP. The ATP sulfurylase reaction is extremely unfavorable with a Keq estimated at 1 x 10-8 and so different mechanisms have evolved to promote APS formation. In Escherichia coli APS formation is coupled to GTP hydrolysis. In higher eukaryotes, ATP sulfurylase and APS kinase are fused on a single polypeptide and APS synthesis is promoted by channeling APS from the ATP sulfurylase active site into the APS kinase active site. We solved the structure of ATP sulfurylase from Saccharomyces cerevisiae. This enzyme adopts a homohexameric arrangement. The C-terminal domain of each monomer though catalytically inactive is structurally very similar to APS kinase and a surface groove was noted that could act as a substrate channel between the ATP sulfurylase and APS kinase-like domains of the enzyme. Although it would appear that this enzyme is evolutionarily related to a bifunctional enzyme how APS formation is promoted is unclear as the APS kinase domain is catalytically defunct. Using yeast two-hybrid analysis we plan to examine if the yeast ATP sulfurylase interacts with other proteins to promote the APS synthesis reaction.