Cohen R E, Schachman H K
J Biol Chem. 1986 Feb 25;261(6):2623-31.
The binding of the bisubstrate ligand N-(phosphonacetyl)-L-aspartate (PALA) to the active sites of both the free catalytic subunit of aspartate transcarbamoylase and the intact holoenzyme causes conformational changes which have been studied extensively. However, no kinetic information has been available about the sequence of events occurring during the formation or dissociation of the complexes. Stopped flow kinetics, 31P saturation transfer NMR spectroscopy, and presteady-state kinetics were used to monitor the interaction of PALA with the catalytic subunit (or a derivative containing nitrotyrosyl chromophores which served as spectral probes). The various experimental approaches lead to a mechanism that includes a rapid binding of PALA with an "on" rate of about 10(8)M-1s-1 and an "off" rate of 28 s-1, followed by a much slower isomerization of the complex with a forward rate constant of 0.18 s-1. Analysis of the presteady-state bursts of enzyme activity when the protein is added to a mixture of substrates and PALA and of the lag in activity when the PALA complex with catalytic subunit is added to substrates yielded a rate constant for the reverse isomerization of 0.018s-1. Thus, the conformational change subsequent to PALA binding leads to a 10-fold increase in the equilibrium constant for complex formation. Stopped flow kinetic measurements of the spectral change resulting from mixing the complex of PALA and nitrated protein with native enzyme showed a slow process with a t1/2 of about 11 s, whereas 31P saturation transfer NMR experiments yielded at t1/2 of about 260 ms for the dissociation of PALA from the complex. This apparent disparity is understood in terms of the two-step binding scheme where rapid dissociation of the initial ligand X enzyme complex is measured by the NMR technique and the slow isomerization of the complex is responsible for the bulk of the stopped flow signal.
双底物配体N-（膦酰乙酰基）-L-天冬氨酸（PALA）与天冬氨酸转氨甲酰酶的游离催化亚基及完整全酶的活性位点结合会引起构象变化，对此已进行了广泛研究。然而，关于复合物形成或解离过程中发生的一系列事件，尚无动力学信息。采用停流动力学、31P饱和转移核磁共振光谱法和稳态前动力学来监测PALA与催化亚基（或含有硝基酪氨酸发色团作为光谱探针的衍生物）的相互作用。各种实验方法得出了一种机制，该机制包括PALA的快速结合，其“结合”速率约为10(8)M-1s-1，“解离”速率为28 s-1，随后复合物发生慢得多的异构化，正向速率常数为0.18 s-1。当将蛋白质加入底物和PALA的混合物中时，对酶活性的稳态前爆发进行分析，以及当将PALA与催化亚基的复合物加入底物中时对活性滞后情况进行分析，得出反向异构化的速率常数为0.018s-1。因此，PALA结合后的构象变化导致复合物形成的平衡常数增加了10倍。将PALA与硝化蛋白质的复合物与天然酶混合所产生的光谱变化的停流动力学测量显示，这是一个缓慢的过程，t1/2约为11 s，而31P饱和转移核磁共振实验得出PALA从复合物中解离的t1/2约为260 ms。根据两步结合方案可以理解这种明显的差异，其中初始配体X酶复合物的快速解离由核磁共振技术测量，而复合物的缓慢异构化是停流信号的主要来源。
