Epizyme, Cambridge, MA 02139, USA.
Anal Biochem. 2011 Sep 15;416(2):206-10. doi: 10.1016/j.ab.2011.05.029. Epub 2011 May 27.
Jump dilution analysis is commonly used to evaluate the reversibility of inhibition and to quantify the residence time of the inhibitor-enzyme complex. During hit and lead characterization, one sometimes observes apparently linear progress curves after jump dilution that display activity recoveries that are intermediate between those expected for fully reversible and irreversible inhibition. Computer simulations of progress curves after jump dilution indicate that seemingly linear progress curves can result when dealing with tight-binding inhibitors if substoichiometric concentrations of inhibitor are preincubated with enzyme. In this situation, the activity recovered is comparable to that expected for instantaneously reversible inhibitors. In addition, simulations demonstrate that intermediate values of activity recovery may be observed for compounds with modestly slow dissociation rates (i.e., residence times >0min but ≤20min) when the attending curvature of the data is not accounted for. The observation of intermediate values of recovery can, thus, serve as an indication of either modest residence time or a contaminating inactivator within an inhibitor sample, in either case prompting greater scrutiny of the test compound.
跳变稀释分析通常用于评估抑制作用的可逆性，并定量测定抑制剂-酶复合物的停留时间。在命中和先导化合物表征过程中，有时会在跳变稀释后观察到明显的线性进展曲线，这些曲线显示的活性恢复介于完全可逆和不可逆抑制之间。跳变稀释后进展曲线的计算机模拟表明，如果以亚化学计量浓度的抑制剂预先孵育酶，则在处理紧密结合抑制剂时可能会出现看似线性的进展曲线。在这种情况下，恢复的活性与预期的瞬时可逆抑制剂相当。此外，模拟还表明，当数据的曲率未被考虑时，对于解离速率适中（即停留时间>0min 但≤20min）的化合物，可能会观察到中等活性恢复值。因此，恢复值的中间值观察结果可作为抑制剂样品中停留时间适中或存在污染失活剂的指示，无论哪种情况都促使对测试化合物进行更仔细的检查。
