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

K. Venkatachalam,H. Akita,C. Strott

Published 1998 in Journal of Biological Chemistry

ABSTRACT

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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