AI Article Synopsis
- Stopped-flow fluorescence spectroscopy is an advanced technique that allows for the precise measurement of rapid enzyme reactions by detecting changes in fluorescence.
- A variety of fluorophores can be used, enabling the observation of binding events, structural changes, and catalytic activity, which helps to explore the dynamic nature of reactions fully.
- The chapter focuses on ribonuclease P as a case study to determine kinetic constants related to substrate interactions, helping to address mechanistic inquiries about factors like reaction conditions and mutations.
Article 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.
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