3-Phosphoadenosine-5-phosphosulfate (PAPS) synthase is a bifunctional protein consisting of an NH 2 -terminal APS kinase and a COOH-terminal ATP sulfurylase. Both catalytic activities require ATP; the APS kinase domain involves cleavage of the -␥ phosphodiester bond of ATP, whereas the ATP sulfurylase domain involves cleavage of the ␣- phosphodiester bond of ATP. Previous mutational studies have suggested that -␥ phosphodiesterase activity involves a highly conserved NTP-binding P-loop motif located in the adenosine-5-phosphosulfate kinase domain of PAPS synthases. Sequence alignment analysis of PAPS synthases and the superfamily of TagD-related nucleotidylyltransferases revealed the presence of a highly conserved HXGH motif in the ATP sulfurylase domain of PAPS synthases, a motif implicated in the ␣- phosphodiesterase activity of cytidylyltransferases. Thus, site-selected mutagenesis of the HXGH motif in the ATP sulfurylase domain of human PAPS synthase (amino acids 425-428) was performed to examine this possibility. Either H425A or H428A mutation produced an inactive enzyme. In contrast, a N426K mutation resulted in increased enzymatic activity. A G427A single mutant resulted in only a modest 30% reduction in catalytic activity, whereas a G427A/H428A double mutant produced an inactive enzyme. These results suggest an important role for the HXGH histidines in the ATP sulfurylase activity of bifunctional PAPS synthase and support the hypothesis that the highly conserved HXGH motif found in the ATP sulfurylase domain of PAPS synthases is involved in ATP binding and ␣- phosphodiesterase activity.In the course of sulfonation, inorganic sulfate must be activated prior to being transferred to an acceptor molecule (1), and the activated sulfate molecule is 3Ј-phosphoadenosine 5Ј-phosphosulfate (PAPS).1 The activation of inorganic sulfate to form PAPS results from the concerted action of two enzymes (1). The first step is catalyzed by ATP sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4) and involves the reaction of inorganic sulfate with ATP to form adenosine 5Ј-phosphosulfate (APS) and inorganic pyrophosphate. This reaction results in the formation of a high energy phosphoric-sulfuric acid anhydride bond that is the chemical basis for sulfate activation (2). The second step involves APS reacting with another molecule of ATP to form PAPS and ADP and is catalyzed by APS kinase (ATP:adenylylsulfate 3Ј-phosphotransferase, EC 2.7.1.25). ATP sulfurylase and APS kinase cloned from bacteria, Fungi, yeast, and plants are found on separate polypeptide chains; however, in mammalian species, the marine worm, and the fruit fly, gene fusion has occurred, and the two enzymes are integral to bifunctional PAPS synthase. It has been clearly established that the NH 2 -terminal region of human (h) PAPS synthase constitutes the APS kinase domain, whereas the ATP sulfurylase domain is located in the COOH-terminal portion of this bifunctional protein (3). That is, the NH 2 -terminal 268 amino acids of hPAPS synthase (623 amino...