Energy Coupling Nicotinate Phosphoribosyltransferase

烟酸磷酸核糖基转移酶在形成NAMN的每个周期中水解1分子ATP，并且这两个过程是能量耦合的。本文提出了一种新的交替亲和力机制，在这种偶联中，ATP的使用产生了对磷酸化核糖基转移酶反应底物具有更高亲和力的E-P；磷酸核糖基转移后，产物焦磷酸盐引发的E- p强制水解再生低亲和力的E形式并导致产物释放。一个E-P被证明是机械参与偶联，ADP/ATP交换和蛋白质化学将用于表征它。在磷酸化残基上诱变将使atp酶和NAMN合成酶的活性解偶联。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形成过程中的变化。快速淬火研究将允许确定E-P水解的顺序和磷酸化核糖基转移的机制。通过了解NAPRTase的能量耦合机制。这将显示亲和性的变化如何导致更复杂的系统（如ca - atp酶和其他载体转运蛋白）中的耦合。

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.