Measurement of nucleotide hydrolysis using fluorescent biosensors for phosphate

Abstract

Assays for the detection of inorganic phosphate (Pi) are widely used to measure the activity of nucleotide hydrolyzing enzymes, such as ATPases and GTPases. The fluorescent biosensors for Pi, described here, are based on fluorescently labeled versions of E. coli phosphate-binding protein (PBP), which translates Pi binding into a large change in fluorescence intensity. In comparison with other Pi-detection systems, these biosensors are characterized by a high sensitivity (sub-micromolar Pi 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 Pi 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, Km and kcat, of the ATP-dependent chromatin remodeler Chd1, in a plate reader format. In addition, the measurement of Pi 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.