Abstract:
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.
摘要:
停流荧光光谱法是一种用于测量快速酶动力学的高灵敏度方法。可以使用广泛的荧光团,可以测量荧光和荧光偏振。因此,绑定,构象变化,催化可以,原则上,被测量,使其有助于探测反应的整个动力学景观。在这一章中,我们使用细菌RNA加工酶核糖核酸酶P(RNaseP)作为模型系统来说明底物结合和裂解的动力学常数的测定,因此允许有关反应条件影响的机械问题,突变,或药物结合需要回答。