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J Biol Chem, Vol. 273, Issue 30, 19311-19320, July 24, 1998

Molecular Cloning, Expression, and Characterization of Human Bifunctional 3'-Phosphoadenosine 5'-Phosphosulfate Synthase and Its Functional Domains

From the Section on Steroid Regulation, Endocrinology, and Reproduction Research Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892-4510

The universal sulfonate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), is synthesized by the concerted action of ATP sulfurylase and adenosine 5'-phosphosulfate (APS) kinase, which in animals are fused into a bifunctional protein. The cDNA for human PAPS synthase (hPAPSS) along with polymerase chain reaction products corresponding to several NH₂- and COOH-terminal fragments were cloned and expressed in COS-1 cells. A 1-268-amino acid fragment expressed APS kinase activity, whereas a 220-623 fragment evinced ATP sulfurylase activity. The 1-268 fragment and full-length hPAPSS (1-623) exhibited hyperbolic responses against APS substrate with equivalent K_m values (0.6 and 0.4 µM, respectively). The 1-268 fragment demonstrated Michaelis-Menten kinetics against ATP as substrate (K_m 0.26 mM); however, full-length hPAPSS exhibited a sigmoidal response (apparent K_m 1.5 mM) suggesting cooperative binding. Catalytic efficiency (V_max/K_m) of the 1-268 fragment was 64-fold higher than full-length hPAPSS for ATP. The kinetic data suggest that the COOH-terminal domain of hPAPSS exerts a regulatory role over APS kinase activity located in the NH₂-terminal domain of this bifunctional protein. In addition, the 1-268 fragment and full-length hPAPSS were overexpressed in Escherichia coli and column purified. Purified full-length hPAPSS, in contrast to the COS-1 cell-expressed cDNA construct, exhibited a hyperbolic response curve against ATP suggesting that hPAPSS is perhaps modified in vivo.

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