Department of Chemical and Biomedical Engineering, University of Missouri, Columbia, MO, USA.
Department of Nutrition, Dietetics and Food Sciences, Utah State University, Logan, UT, USA.
Sci Rep. 2024 Sep 11;14(1):21192. doi: 10.1038/s41598-024-72028-4.
A key player in energy metabolism is phosphofructokinase-1 (PFK1) whose activity and behavior strongly influence glycolysis and thus have implications in many areas. In this research, PFK1 assays were performed to convert F6P and ATP into F-1,6-P and ADP for varied pH and ATP concentrations. PFK1 activity was assessed by evaluating F-1,6-P generation velocity in two ways: (1) directly calculating the time slope from the first two or more datapoints of measured product concentration (the initial-velocity method), and (2) by fitting all the datapoints with a differential equation explicitly representing the effects of ATP and pH (the modeling method). Similar general trends of inhibition were shown by both methods, but the former gives only a qualitative picture while the modeling method yields the degree of inhibition because the model can separate the two simultaneous roles of ATP as both a substrate of reaction and an inhibitor of PFK1. Analysis based on the model suggests that the ATP affinity is much greater to the PFK1 catalytic site than to the inhibitory site, but the inhibited ATP-PFK1-ATP complex is much slower than the uninhibited PFK1-ATP complex in product generation, leading to reduced overall reaction velocity when ATP concentration increases. The initial-velocity method is simple and useful for general observation of enzyme activity while the modeling method has advantages in quantifying the inhibition effects and providing insights into the process.
在能量代谢中，磷酸果糖激酶-1（PFK1）是一个关键的调节因子，其活性和行为强烈影响糖酵解，因此在许多领域都有重要意义。在这项研究中，我们进行了 PFK1 测定，以将 F6P 和 ATP 转化为 F-1,6-P 和 ADP，实验条件为不同的 pH 和 ATP 浓度。通过两种方法评估 F-1,6-P 的生成速度来评估 PFK1 活性：（1）直接从测量产物浓度的前两个或更多数据点计算时间斜率（初始速度法），以及（2）通过拟合所有数据点，使用明确表示 ATP 和 pH 影响的微分方程（建模法）。两种方法都显示出相似的抑制趋势，但前者只能提供定性的结果，而建模方法可以提供抑制程度，因为该模型可以将 ATP 作为反应底物和 PFK1 抑制剂的两个同时作用分开。基于模型的分析表明，ATP 对 PFK1 催化位点的亲和力远大于对抑制位点的亲和力，但与未受抑制的 PFK1-ATP 复合物相比，受抑制的 ATP-PFK1-ATP 复合物在产物生成中的速度要慢得多，当 ATP 浓度增加时，导致整体反应速度降低。初始速度法简单且可用于观察酶活性的一般情况，而建模法则具有量化抑制效果和深入了解反应过程的优势。
