Role of aspartic acid residues D87 and D89 in APS kinase domain of human 3'-phosphoadenosine 5'-phosphosulfate synthase 1 and 2b: A commonality with phosphatases/kinases.

3'-phosphoadenosine 5'-phosphosulfate (PAPS) is synthesized in two steps by PAPS synthase (PAPSS). PAPSS is comprised of ATP sulfurylase (ATPS) and APS kinase (APSK) domain activities. ATPS combines inorganic sulfate with α-phosphoryl of ATP to form adenosine 5'-phosphosulfate (APS) and PPi. In the second step APS is phosphorylated at 3'-OH using another mole of ATP to form PAPS and ADP catalyzed by APSK. The transfer of gamma-phosphoryl from ATP onto 3'-OH requires Mg and purported to involve residues DGDN. We report that mutation of either aspartic residue to alanine completely abolishes APSK activity in PAPS formation. PAPSS is an, unique enzyme that binds to four different nucleotides: ATP and APS on both ATPS and APSK domains and ADP and PAPS exclusively on the APSK domain. The thermodynamic binding and the catalytic interplay must be very tightly controlled to form the end-product PAPS in the forward direction. Though APS binds to ATPS and APSK, in ATPS domain, the APS is a product and for APSK it is a substrate. DGDN motif is absent in ATPS and present in APSK. Mutation of D and D did not hamper ATPS activity however abolished APSK activity severely. Thus, DGDN region is required for stabilization of Mg-ATP, in the process of splitting the γ-phosphoryl from ATP and transfer of γ-phosphoryl onto 3'-OH of APS to form PAPS a process that cannot be achieved by ATPS domain. In addition, gammaP-ATP, trapped phosphoryl enzyme intermediate more with PAPSS2 than with PAPSS1. This suggests inherent active site residues could control novel catalytic differences. Molecular docking studies of hPAPSS1with ATP + Mg and APS of wild type and mutants supports the experimental results.