restriction factor SAMHD1 [56]. By coupling the reaction with
yeast exopolyphosphatase (PPX1), the primary product, triphosphate, is converted into pyrophosphate (PP i ) and P i , which is then
detected by MDCC-PBP. Similarly, the combination of inorganic
pyrophosphatase (PPase) and MDCC-PBP can be applied to measure the formation of PP i in a number of enzymatic reactions. This
method has been used, for example, to measure PP i release from
protein prenyltransferases [57] or DNA and RNA polymerases
[58–61]. Finally, real-time kinetics of nuclease reactions were
measured using MDCC-PBP in combination with alkaline phosphatase or T4 polynucleotide kinase as secondary enzyme, which
cleave off the terminal phosphate from the newly generated nucleic
acid products [62].
The key to successfully applying these coupled-enzyme assays is
that the reaction of the secondary enzyme is much faster than the
reaction under study and therefore is not rate-limiting. This is
particularly critical under pre-steady state conditions and requires
careful experimental design and controls, see for example [59].
3.4 Summary
Fluorescent biosensors for phosphate, MDCC-PBP, and Rho-PBP
are very useful tools to measure the kinetics of nucleotide hydrolysis
by enzymes, such as ATPases and GTPases. The assay principle is
simple and is based on direct detection of the product, so apart
from product depletion, the reaction being studied is not modified.
The biosensors allow real-time measurements of P i release at high
time resolution, down to milliseconds. Other P i detecting systems
are much more limited, having response times of seconds (coupled
enzyme assays) or even minutes (malachite green), so the latter can
only be used in a discontinuous format.
The ability to measure P i release in real time has greatly added
to our understanding of the mechanism of motility proteins like
myosin, kinesin, or DNA helicases. In addition, due to the simplicity, the P i biosensor assays have also found application in screening
for inhibitors. It will be interesting to see how the usage of phosphate biosensors will be extended in the future, for example, in the
fields of single-molecule enzymology or drug discovery.
4 Notes
1. In this chapter, all assays are described using MDCC-PBP,
which is the original biosensor variant, is easier to prepare,
and therefore is most commonly used for steady state and
pre-steady state kinetics. However, the tetramethylrhodamine
version, Rho-PBP, can be used interchangeably in most assay
types, just by adjusting the excitation and detection wavelengths (excitation maximum 553 nm, emission maximum
575 nm [6]).
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Simone Kunzelmann
yeast exopolyphosphatase (PPX1), the primary product, triphosphate, is converted into pyrophosphate (PP i ) and P i , which is then
detected by MDCC-PBP. Similarly, the combination of inorganic
pyrophosphatase (PPase) and MDCC-PBP can be applied to measure the formation of PP i in a number of enzymatic reactions. This
method has been used, for example, to measure PP i release from
protein prenyltransferases [57] or DNA and RNA polymerases
[58–61]. Finally, real-time kinetics of nuclease reactions were
measured using MDCC-PBP in combination with alkaline phosphatase or T4 polynucleotide kinase as secondary enzyme, which
cleave off the terminal phosphate from the newly generated nucleic
acid products [62].
The key to successfully applying these coupled-enzyme assays is
that the reaction of the secondary enzyme is much faster than the
reaction under study and therefore is not rate-limiting. This is
particularly critical under pre-steady state conditions and requires
careful experimental design and controls, see for example [59].
3.4 Summary
Fluorescent biosensors for phosphate, MDCC-PBP, and Rho-PBP
are very useful tools to measure the kinetics of nucleotide hydrolysis
by enzymes, such as ATPases and GTPases. The assay principle is
simple and is based on direct detection of the product, so apart
from product depletion, the reaction being studied is not modified.
The biosensors allow real-time measurements of P i release at high
time resolution, down to milliseconds. Other P i detecting systems
are much more limited, having response times of seconds (coupled
enzyme assays) or even minutes (malachite green), so the latter can
only be used in a discontinuous format.
The ability to measure P i release in real time has greatly added
to our understanding of the mechanism of motility proteins like
myosin, kinesin, or DNA helicases. In addition, due to the simplicity, the P i biosensor assays have also found application in screening
for inhibitors. It will be interesting to see how the usage of phosphate biosensors will be extended in the future, for example, in the
fields of single-molecule enzymology or drug discovery.
4 Notes
1. In this chapter, all assays are described using MDCC-PBP,
which is the original biosensor variant, is easier to prepare,
and therefore is most commonly used for steady state and
pre-steady state kinetics. However, the tetramethylrhodamine
version, Rho-PBP, can be used interchangeably in most assay
types, just by adjusting the excitation and detection wavelengths (excitation maximum 553 nm, emission maximum
575 nm [6]).
312
Simone Kunzelmann