Isolation of a 55 kd protein from the guinea pig adrenal that catalyzes production of the sulfotransferase sulfonate donor molecule 3′-phosphoadenosine 5′-phosphosulfate (PAPS): evidence for intrinsic sulfatase activity

Biological sulfonation carried out by sulfotransferases requires the universal sulfonate donor molecule PAPS. In addition to the activity of sulfotransferases, the availability of PAPS could also be rate-limiting. The activation of inorganic sulfate to form PAPS is the result of the concerted action of two enzymes: ATP sulfurylase (EC 2.7.7.4) and adenosine 5′-phosphosulfate (APS) kinase (EC 2.7.1.25). ATP sulfurylase (Mr 35.2-63.9 kDa) and APS kinase (M, 22.3-29.8 kDa) have been isolated and cloned from bacteria, fungi, yeast and plants; neither enzyme has been cloned from a mammalian source. We isolated ATP sulfurylase and APS kinase from guinea pig adrenals and found that, in contrast to prokaryotes and lower eukaryotes, the enzymes co-purified and resulted in the purification of a 55 kDa protein termed PAPS synthase. Thus, in mammals ATP sulfurylase and APS kinase are physically linked on a single protein. ATP sulfurylase assayed in the thermodynamically-favored reverse direction has a Km of 20 /iM for PPj and 82 nM for APS. APS kinase assayed in the forward direction has a K_m of 50 pM for ATP and 90 nM for APS. Unexpectedly, it was found that isolated guinea pig adrenal PAPS synthase also demonstrated sulfatase activity. The sulfatase activity was ascertained to be ATP-dependent and specific for PAPS. Conclusion: the 55 kDa protein purified to apparent homogeneity from the guinea pig adrenal gland is a multifunctional protein with intrinsic activities for ATP sulfurylase, APS kinase and PAPS sulfatase. The physiological significance of the ATP-dependent, PAPSspedfic sulfatase activity associated with PAPS synthase remains to be determined.