YEAST TWO-HYBRID ANALYSIS OF ATP SULFURYLASE

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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.