Activity and stability of recombinant bifunctional rearranged and monofunctional domains of ATP-sulfurylase and adenosine 5 '-phosphosulfate kinase

Murine adenosine 3'-phosphate 5'-phosphosulfate (PAPS) synthetase consists of a COOH-terminal ATP-sulfurylase domain covalently linked through a nonho mologous intervening sequence to an NH2-terminal adenosine 5'-phosphosulfat e (APS) kinase domain forming a bifunctional fused protein. Possible advant ages of bifunctionality were probed by separating the domains on the cDNA l evel and expressing them as monofunctional proteins. Expressed protein gene rated from the ATP-sulfurylase domain alone was fully active in both the fo rward and reverse sulfurylase assays. APS kinase-only recombinants exhibite d no kinase activity. However, extension of the kinase domain at the COOH t erminus by inclusion of the 36 residue linker region restored kinase activi ty. An equimolar mixture of the two monofunctional enzymes catalyzed the ov erall reaction (synthesis of PAPS from ATP + SO42-) comparably to the fused bifunctional enzyme. The importance of the domain order and organization w as demonstrated by generation of a series of rearranged recombinants in whi ch the order of the two active domains was reversed or altered relative to the linker region, The critical role of the linker region was established b y generation of recombinants that had the linker deleted or rearranged rela tive to the two active domains. The intrinsic stability of the various reco mbinants was also investigated by measuring enzyme deactivation as a functi on of time of incubation at 25 or 37 degrees C, The expressed monofunctiona l ATP-sulfurylase, which was initially fully active, was unstable compared with the fused bifunctional wild type enzyme, decaying with a t(1/2) of 10 min at 37 degrees C, Progressive extension by addition of kinase sequence a t the NH2-terminal side of the sulfurylase recombinant eventually stabilize d sulfurylase activity. Sulfurylase activity was significantly destabilized in a time-dependent manner in the rearranged proteins as well. In contrast , no significant deactivation of any truncated kinase-containing recombinan ts or misordered kinase recombinants was observed at either temperature. It would therefore appear that fusion of the two enzymes enhances the intrins ic stability of the sulfurylase only.