MacKerell A D, Rigler R, Hahn U, Saenger W
Department of Medical Biophysics, Karolinska Institutet, Stockholm, Sweden.
Biochim Biophys Acta. 1991 Mar 4;1073(2):357-65. doi: 10.1016/0304-4165(91)90143-5.
Fluorescence titrations and temperature-jump relaxation experiments were performed as a function of temperature on ribonuclease T1 with the inhibitors 2'GMP and 3'GMP to obtain information on the energetics and molecular events controlling the binding of those inhibitors. Results from the titration and temperature-jump experiments were in agreement concerning the equilibrium constant. The larger equilibrium constant for 2'GMP is enthalpic in origin and is due to both a higher on rate and a lower off rate as compared to 3'GMP. On rates for both inhibitors appear to be below the diffusion controlled limit, apparently due to conformational changes in the portion of the active site responsible for recognition of the guanine base. Comparison of the measured enthalpic and entropic terms associated with the equilibrium constant determined from the fluorescence titrations are in disagreement with those calculated from the on and off rates indicating the presence of an induced conformational change in the 2'GMP-enzyme complex. This second conformational change appears to be due to additional interactions between 2'GMP and the catalytic portion of the active site, which may also be responsible for the differences in the binding constant, the on rate and the off rate between 2'GMP and 3'GMP.
以核糖核酸酶T1为研究对象，使用抑制剂2'-鸟苷酸（2'GMP）和3'-鸟苷酸（3'GMP），在不同温度下进行荧光滴定和温度跳跃弛豫实验，以获取有关控制这些抑制剂结合的能量学和分子事件的信息。滴定实验和温度跳跃实验所得结果在平衡常数方面是一致的。2'GMP的平衡常数较大，其起源是焓驱动的，这是由于与3'GMP相比，它具有更高的结合速率和更低的解离速率。两种抑制剂的结合速率似乎都低于扩散控制极限，这显然是由于活性位点中负责识别鸟嘌呤碱基的部分发生了构象变化。通过荧光滴定确定的与平衡常数相关的测量焓项和熵项与根据结合和解离速率计算出的结果不一致，这表明2'GMP-酶复合物中存在诱导的构象变化。这种第二种构象变化似乎是由于2'GMP与活性位点催化部分之间的额外相互作用引起的，这也可能是2'GMP和3'GMP之间结合常数、结合速率和解离速率存在差异的原因。
