Department of Chemistry, University of Florida, Gainesville, FL, USA.
Methods Mol Biol. 2024;2822:431-441. doi: 10.1007/978-1-0716-3918-4_27.
Stopped-flow fluorescence spectroscopy is a highly sensitive method for measuring rapid enzyme kinetics. A wide range of fluorophores can be employed, and fluorescence and fluorescence polarization can be measured. Thus, binding, conformational changes, and catalysis can, in principle, be measured, making it helpful in probing the entire kinetic landscape of a reaction. In this chapter, we use the bacterial RNA processing enzyme ribonuclease P (RNase P) as a model system to illustrate the determination of the kinetic constants for substrate binding and cleavage, thus allowing mechanistic questions regarding the effects of reaction conditions, mutations, or drug binding to be answered.
停流荧光光谱法是一种测量快速酶动力学的高灵敏度方法。可以使用多种荧光团，并可以测量荧光和荧光偏振。因此，原则上可以测量结合、构象变化和催化作用，这有助于探测反应的整个动力学景观。在本章中，我们使用细菌 RNA 加工酶核糖核酸酶 P（RNase P）作为模型系统，说明底物结合和切割的动力学常数的确定，从而可以回答关于反应条件、突变或药物结合影响的机制问题。
