Structural Biology Science Technology Platform, The Francis Crick Institute, London, UK.
Methods Mol Biol. 2021;2263:289-318. doi: 10.1007/978-1-0716-1197-5_13.
Assays for the detection of inorganic phosphate (P) are widely used to measure the activity of nucleotide hydrolyzing enzymes, such as ATPases and GTPases. The fluorescent biosensors for P, described here, are based on fluorescently labeled versions of E. coli phosphate-binding protein (PBP), which translates P binding into a large change in fluorescence intensity. In comparison with other P-detection systems, these biosensors are characterized by a high sensitivity (sub-micromolar P concentrations) and high time resolution (tens of milliseconds), and they are therefore particularly well suited for measurements of phosphate ester hydrolysis in real time. In this chapter, it is described how the P biosensors can be used to measure kinetics of ATPase and GTPase reactions, both under steady state and pre-steady state conditions. An example protocol is given for determining steady state kinetic parameters, K and k, of the ATP-dependent chromatin remodeler Chd1, in a plate reader format. In addition, the measurement of P release kinetics under pre-steady state conditions is described, including a detailed experimental procedure for a single turnover measurement of ATP hydrolysis by the ABC-type ATPase SufBC using rapid mixing.
用于检测无机磷酸盐 (P) 的测定法被广泛用于测量核苷酸水解酶（如 ATP 酶和 GTP 酶）的活性。此处描述的用于 P 的荧光生物传感器基于大肠杆菌磷酸盐结合蛋白 (PBP) 的荧光标记版本，其将 P 结合转化为荧光强度的大幅变化。与其他 P 检测系统相比，这些生物传感器具有高灵敏度（亚微米级 P 浓度）和高时间分辨率（数十毫秒）的特点，因此特别适合实时测量磷酸酯水解。在本章中，将描述如何使用 P 生物传感器来测量 ATP 酶和 GTP 酶反应的动力学，包括稳态和准稳态条件下的动力学。给出了一个使用板式读取器确定依赖于 ATP 的染色质重塑酶 Chd1 的稳态动力学参数 K 和 k 的示例方案。此外，还描述了在准稳态条件下测量 P 释放动力学的方法，包括使用快速混合对 ABC 型 ATP 酶 SufBC 的 ATP 水解进行单次测量的详细实验程序。
