Molecular cloning, expression, and characterization of human bifunctional 3'-phosphoadenosine 5'-phosphosulfate synthase and its functional domains.
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 Km values (0.6 and 0.4 microM, respectively). The 1-268 fragment demonstrated Michaelis-Menten kinetics against ATP as substrate (Km 0.26 mM); however, full-length hPAPSS exhibited a sigmoidal response (apparent Km 1.5 mM) suggesting cooperative binding. Catalytic efficiency (Vmax/Km) 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.
Venkatachalam
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KV
Section on Steroid Regulation, Endocrinology, and Reproduction Research Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892-4510, USA.
Akita
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H
Strott
C A
CA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D002384:Catalysis; D003001:Cloning, Molecular; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D013430:Sulfate Adenylyltransferase / Q000235:genetics* / Q000378:metabolism
D009097:Multienzyme Complexes; C022322:PAPS synthetase; D013430:Sulfate Adenylyltransferase