Energy Coupling Nicotinate Phosphoribosyltransferase

烟酸磷酸核糖基转移酶在NAMN形成的每个循环中水解1个ATP分子，这两个过程是能量耦合的。对于这种偶联，提出了一种新的交替亲和力机制，其中利用ATP生成与磷酸核糖转移酶反应底物亲和力增加的E-P；在磷酸核糖转移后，由产物焦磷酸触发的强制E-P水解会重新生成低亲和力的E形式并导致产物释放。E-P参与了偶联，ADP/ATP交换和蛋白质化学将被用来表征它。在磷酸化残基上的突变将允许解偶联该酶的ATPase和NAMN合成活性。将进行立体化学研究，以确定是否存在其他磷酸化中间体。与E相比，E-P显著降低了底物PRPP和烟酸盐的Km值，进一步的结合研究将量化E-P形成过程中发生的变化。快速猝灭研究将使确定E-P水解和磷酸核糖基团转移的顺序成为可能。通过了解NAPRTase中的能量偶联机制。这将展示亲和力的变化如何导致更复杂的系统中的偶联，如Ca-ATPase和其他载体转运蛋白。

Nicotinate phosphoribosyltransferase hydrolyzes 1 molecule of ATP for each cycle of NAMN formation, and the two processes are energetically coupled. A novel alternating affinity mechanism is proposed for this coupling in which ATP use generates an E-P with increased affinity for the substrates of the phosphoribosyltransferase reaction; after phosphoribosyl group transfer, compulsory E-P hydrolysis triggered by product pyrophosphate regenerates the low affinity E form and results in product release. An E-P was shown to be mechanistically involved in coupling, ADP/ATP exchanges and protein chemistry will be used to characterize it. Mutagenesis at the phosphorylated residue will allow uncoupling the ATPase and NAMN synthesis activities of the enzyme. Stereochemical studies will be carried out to determine if other phosphorylated intermediates exist. The E-P has dramatically decreased Km values for substrates PRPP and nicotinate, compared to E, and the use of further binding studies will quantitate the changes that occur on E-P formation. Rapid quench studies will allow the determination the order of E-P hydrolysis and phosphoribosyl group transfer in the mechanism. By learning the mechanism of energy coupling in NAPRTase. This will show how alternation in affinity can lead to coupling in more complex systems such Ca-ATPase and other vectorial transporters.