Chapter 13
Measurement of Nucleotide Hydrolysis Using Fluorescent
Biosensors for Phosphate
Simone Kunzelmann
Abstract
Assays for the detection of inorganic phosphate (P i ) are widely used to measure the activity of nucleotide
hydrolyzing enzymes, such as ATPases and GTPases. The fluorescent biosensors for P i , described here, are
based on fluorescently labeled versions of E. coli phosphate-binding protein (PBP), which translates P i
binding into a large change in fluorescence intensity. In comparison with other P i -detection systems, these
biosensors are characterized by a high sensitivity (sub-micromolar P i 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 i 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 m and k cat , of the
ATP-dependent chromatin remodeler Chd1, in a plate reader format. In addition, the measurement of P i
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.
Key words Phosphate detection, Kinetics, ATPase, GTPase, Phosphate release, Enzyme mechanism,
Single turnover, Multi-turnover, Stopped-flow
1 Introduction
Inorganic phosphate is the product of numerous cellular reactions
catalyzed by enzymes called phosphohydrolases. These include
several families of nucleoside triphosphatases, e.g., ATPases and
GTPases, and different types of phosphatases that cleave inorganic
phosphate (P i ) from nucleic acid, protein, or small metabolite substrates. Fluorescent phosphate-binding protein is widely used as a
probe to monitor P i release from these reactions in real time.
A fluorescent version of the Escherichia coli phosphate-binding
protein (MDCC-PBP) was first developed as a P i biosensor in the
laboratory of Martin Webb [1], initially with the aim to study the
role of P i release from myosin in muscle fibers [2, 3]. MDCC-PBP
consists of a single cysteine mutant of periplasmic
Tina Daviter et al. (eds.), Protein-Ligand Interactions: Methods and Applications, Methods in Molecular Biology, vol. 2263,
https://doi.org/10.1007/978-1-0716-1197-5_13, © Springer Science+Business Media, LLC, part of Springer Nature 2021
289
Measurement of Nucleotide Hydrolysis Using Fluorescent
Biosensors for Phosphate
Simone Kunzelmann
Abstract
Assays for the detection of inorganic phosphate (P i ) are widely used to measure the activity of nucleotide
hydrolyzing enzymes, such as ATPases and GTPases. The fluorescent biosensors for P i , described here, are
based on fluorescently labeled versions of E. coli phosphate-binding protein (PBP), which translates P i
binding into a large change in fluorescence intensity. In comparison with other P i -detection systems, these
biosensors are characterized by a high sensitivity (sub-micromolar P i 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 i 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 m and k cat , of the
ATP-dependent chromatin remodeler Chd1, in a plate reader format. In addition, the measurement of P i
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.
Key words Phosphate detection, Kinetics, ATPase, GTPase, Phosphate release, Enzyme mechanism,
Single turnover, Multi-turnover, Stopped-flow
1 Introduction
Inorganic phosphate is the product of numerous cellular reactions
catalyzed by enzymes called phosphohydrolases. These include
several families of nucleoside triphosphatases, e.g., ATPases and
GTPases, and different types of phosphatases that cleave inorganic
phosphate (P i ) from nucleic acid, protein, or small metabolite substrates. Fluorescent phosphate-binding protein is widely used as a
probe to monitor P i release from these reactions in real time.
A fluorescent version of the Escherichia coli phosphate-binding
protein (MDCC-PBP) was first developed as a P i biosensor in the
laboratory of Martin Webb [1], initially with the aim to study the
role of P i release from myosin in muscle fibers [2, 3]. MDCC-PBP
consists of a single cysteine mutant of periplasmic
Tina Daviter et al. (eds.), Protein-Ligand Interactions: Methods and Applications, Methods in Molecular Biology, vol. 2263,
https://doi.org/10.1007/978-1-0716-1197-5_13, © Springer Science+Business Media, LLC, part of Springer Nature 2021
289