Deyrup A T, Krishnan S, Singh B, Schwartz N B
Departments of Pediatrics and Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois, 60637, USA.
J Biol Chem. 1999 Apr 16;274(16):10751-7. doi: 10.1074/jbc.274.16.10751.
Murine adenosine 3'-phosphate 5'-phosphosulfate (PAPS) synthetase consists of a COOH-terminal ATP-sulfurylase domain covalently linked through a nonhomologous intervening sequence to an NH2-terminal adenosine 5'-phosphosulfate (APS) kinase domain forming a bifunctional fused protein. Possible advantages of bifunctionality were probed by separating the domains on the cDNA level and expressing them as monofunctional proteins. Expressed protein generated from the ATP-sulfurylase domain alone was fully active in both the forward and reverse sulfurylase assays. APS kinase-only recombinants exhibited no kinase activity. However, extension of the kinase domain at the COOH terminus by inclusion of the 36 residue linker region restored kinase activity. An equimolar mixture of the two monofunctional enzymes catalyzed the overall reaction (synthesis of PAPS from ATP + SO42-) comparably to the fused bifunctional enzyme. The importance of the domain order and organization was demonstrated by generation of a series of rearranged recombinants in which 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 by generation of recombinants that had the linker deleted or rearranged relative to the two active domains. The intrinsic stability of the various recombinants was also investigated by measuring enzyme deactivation as a function of time of incubation at 25 or 37 degrees C. The expressed monofunctional ATP-sulfurylase, which was initially fully active, was unstable compared with the fused bifunctional wild type enzyme, decaying with a t1/2 of 10 min at 37 degrees C. Progressive extension by addition of kinase sequence at the NH2-terminal side of the sulfurylase recombinant eventually stabilized 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 recombinants or misordered kinase recombinants was observed at either temperature. It would therefore appear that fusion of the two enzymes enhances the intrinsic stability of the sulfurylase only.
小鼠3'-磷酸腺苷5'-磷酸硫酸酯（PAPS）合成酶由一个COOH末端的ATP硫酸化酶结构域组成，该结构域通过一个非同源的中间序列与一个NH2末端的腺苷5'-磷酸硫酸酯（APS）激酶结构域共价连接，形成一个双功能融合蛋白。通过在cDNA水平上分离这些结构域并将它们表达为单功能蛋白，探究了双功能的可能优势。仅由ATP硫酸化酶结构域产生的表达蛋白在正向和反向硫酸化酶测定中均具有完全活性。仅含APS激酶的重组体没有表现出激酶活性。然而，通过包含36个残基的连接区在COOH末端扩展激酶结构域恢复了激酶活性。两种单功能酶的等摩尔混合物催化整个反应（从ATP + SO42-合成PAPS）的效率与融合双功能酶相当。通过产生一系列重排的重组体证明了结构域顺序和组织的重要性，其中两个活性结构域的顺序相对于连接区被颠倒或改变。通过产生相对于两个活性结构域缺失或重排连接区的重组体，确定了连接区的关键作用。还通过测量在25或37摄氏度下孵育时间与酶失活的关系，研究了各种重组体的内在稳定性。最初完全有活性的表达单功能ATP硫酸化酶与融合双功能野生型酶相比不稳定，在37摄氏度下以10分钟的半衰期衰减。通过在硫酸化酶重组体的NH2末端侧添加激酶序列进行逐步扩展最终稳定了硫酸化酶活性。在重排蛋白中，硫酸化酶活性也以时间依赖性方式显著不稳定。相比之下，在任何一个温度下，未观察到任何含截短激酶的重组体或激酶顺序错误的重组体有明显失活。因此，似乎两种酶的融合仅增强了硫酸化酶的内在稳定性。
