Lucius Aaron L, Jezewska Maria J, Roychowdhury Anasuya, Bujalowski Wlodzimierz
Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch at Galveston, 301 University Boulevard, Galveston, Texas 77555-1053, USA.
Biochemistry. 2006 Jun 13;45(23):7217-36. doi: 10.1021/bi051827e.
Kinetics of the nucleotide binding to the strong (S) and weak (W) nucleotide-binding site of the Escherichia coli PriA helicase have been studied using the fluorescence stopped-flow technique. Experiments were performed with TNP-ADP and TNP-ATP analogues. Binding of the ADP analogue to the strong binding site is a four-step sequential reaction: (PriA)S + D (k1)<-->(k(-1)) + (S)1 (k2)<-->(k(-2)) (S)2 (k3)<-->(k(-3)) (S)3 (k4)<-->(k(-4)) (S)4. Association of TNP-ATP proceeds through an analogous three-step mechanism. The first two steps and intermediates are similar for both cofactors. However, the (S)3 intermediate is dramatically different for ADP and ATP analogues. Its emission is close to the emission of the free TNP-ADP, while it is by a factor of approximately 16 larger than the free TNP-ATP fluorescence. Thus, only the ADP analogue passes through an intermediate where it leaves the hydrophobic cleft of the site. This behavior corroborates with the fact that ADP leaves the ATPase site without undergoing a chemical change. The fast bimolecular step and the sequential mechanism indicate that the site is easily accessible to the cofactor, and it does not undergo an adjustment prior to binding. The subsequent step is also fast and stabilizes the complex. Magnesium profoundly affects the population of intermediates. The data indicate that the dominant (S)2 species is a part of the ATP catalytic cycle. ADP analogue binding to the weak nucleotide-binding site proceeds in a simpler two-step mechanism: (PriA)W + D (k1)<-->(k(-1)) (W)1 (k2)<-->(k(-2)) (W)2 with (W)1 being a dominant intermediate both in the presence and in the absence of Mg2+. The results indicate that the weak site is an allosteric control site in the functioning of the PriA helicase.
利用荧光停流技术研究了核苷酸与大肠杆菌PriA解旋酶的强（S）和弱（W）核苷酸结合位点的结合动力学。实验使用了TNP-ADP和TNP-ATP类似物。ADP类似物与强结合位点的结合是一个四步连续反应：（PriA）S + D （k1）<-->(k(-1)) + （S）1 （k2）<-->(k(-2)) （S）2 （k3）<-->(k(-3)) （S）3 （k4）<-->(k(-4)) （S）4。TNP-ATP的结合通过类似的三步机制进行。两种辅因子的前两步和中间体相似。然而，（S）3中间体对于ADP和ATP类似物有显著差异。其发射接近游离TNP-ADP的发射，而比游离TNP-ATP荧光大约强16倍。因此，只有ADP类似物通过一个中间体，在此中间体中它离开位点的疏水裂缝。这种行为与ADP不发生化学变化就离开ATP酶位点的事实相符。快速的双分子步骤和连续机制表明该位点易于被辅因子接近，并且在结合之前不发生调整。随后的步骤也很快并稳定了复合物。镁深刻影响中间体的数量。数据表明占主导的（S）2物种是ATP催化循环的一部分。ADP类似物与弱核苷酸结合位点的结合以更简单的两步机制进行：（PriA）W + D （k'1）<-->(k'(-1)) （W）1 （k'2）<-->(k'(-2)) （W）2，在有和没有Mg2+的情况下，（W）1都是主要中间体。结果表明弱位点是PriA解旋酶功能中的一个变构控制位点。
