Sulfonation is of fundamental importance in the biotransformation of endobiotics as well as exobiotics. Sulfonation plays a primary role in the post-translational modification of numerous structural and membrane constituents as well as secretary proteins that is absolutely essential for normal development and maintenance of health. The sulfonation of low molecular weight compounds plays a crucial role in hormone action, storage and metabolism. The universal sulfonate donor molecule, 3'- phosphoadenosine 5'-phosphosulfate (PAPS), is synthesized by two catalytic reactions, i.e., ATP sulfurylase and adenosine 5'- phosphosulfate (APS) kinase. In contrast to bacteria, fungi, yeast, and plants where the two enzymes are on separate polypeptide chains, in higher eukaryotes, gene fusion has occurred and the two reactions are intrinsic to a single protein (PAPS synthase). We have cloned human and guinea pig PAPS synthase and our preliminary studies with recombinant constructs have demonstrated that APS kinase activity resides within the NH2-terminal domain, while ATP sulfurylase activity is located in the COOH-terminal domain of the protein. We are currently carrying out studies employing site-directed mutagenesis and recombinant constructs to: 1) determine the significance of highly conserved nucleotide binding motifs within the APS kinase domain; 2) examine a conserved FISP sequence present in the APS kinase domain that is thought to function as a phosphorylated intermediate in the phosphorylation of APS to form PAPS; 3) confirm that there is a common overlapping sequence which is essential for the functionality of both ATP sulfurylase and APS kinase as suggested by our preliminary studies; 4) determine if there is an APS binding pocket within the overlapping sequence; 5) evaluate the regulatory influence of the COOH-terminal segment on APS kinase activity located in the NH2-terminal domain of PAPS synthase as suggested by our kinetic analyses of the full-length fusion protein and the active domains produced by recombinant techniques. The fact that PAPS is such a critical biological molecule in mammals makes its production of vital importance. This, in turn, clearly places PAPS synthase in a strategic position. The more that is understood about this intriguing fused protein that regulates the production of PAPS, the more that will be understood about the evolution, biochemistry and biology of the sulfonation process. The reality that sulfurylation reactions are so widespread, that they involve an impressive legion of molecules, both large (e.g. membrane and extracellular structural elements) and small (e.g. hormones and neurotransmitters), coupled with the fact that PAPS is the indispensable and universal sulfonate donor molecule in mammals, it would not be unreasonable to suspect that knocking out the PAPS synthase gene might prove to be lethal. In fact, the human heritable lethal disorder achondrogenesis type 1 cogently supports this conclusion. Thus, to say that a normally functioning PAPS synthase is mandatory for life itself would not be an overstatement.