malachite green. Therefore, they are ideal tools for measuring the
kinetics of enzyme reactions in real time, in steady state as well as
pre-steady state experiments.
Steady state kinetic assays allow determination of k cat , K m , and
the kinetic efficiency k cat /K m , as well as the study of activation and
inhibition of an enzyme. Biosensors with very fast response times,
like PBP, also provide the time resolution needed for pre-steady
state, kinetic experiments using flash photolysis, or stopped-flow
techniques, which can give a more detailed insight into the mechanism of the enzyme reaction.
In this chapter, I describe how to use phosphate-binding protein to measure the kinetics of ATPase and GTPase reactions.
Protocols are given for determining steady state kinetic parameters,
K m and k cat , in a plate reader format in the absence and presence of
an activator. In addition, I describe examples of direct measurement
of P i release under pre-steady state conditions using stopped-flow
and illustrate what information about the enzyme mechanism can
be deduced from this type of data.
2 Materials
2.1 Phosphate
Biosensor
The coumarin version of the biosensor, MDCC-PBP (see Note 1),
is sold as ready-to-use phosphate sensor by Thermo Fisher Scientific (PV4406/7), but the biosensor used for the experiments in
this chapter was purified and labeled in the laboratory, which is a
straight forward procedure. Rho-PBP is not currently available
commercially. The latest expression vectors for both biosensors
are available from Addgene (http://www.addgene.org/): plasmids
pET22b_PstS_1 for MDCC-PBP and pET22b_PstS_2 for
Rho-PBP [9]. Detailed protocols for purification, labeling, and
characterization of the biosensors can also be found on the
Addgene webpage. Note that the expression system has changed
to IPTG induction [9] since the original publications [1, 5, 6].
2.2 Reaction Buffer
The most critical factor for successful assays of P i is to avoid contamination with P i , which is a very widespread impurity (see Note
2).
All buffer components should be analytical grade. Water used
for buffer preparation and rinsing should be used directly from the
distillation apparatus or Milli-Q system without storage to avoid
contamination. We have worked successfully with double-distilled
water, Milli-Q, and commercial water (HPLC grade). Disposable
plasticware is used where possible and everything is rinsed with
P i -free water before use. If glassware cannot be avoided, it is soaked
in 25% HCl overnight and rinsed with water. The pH of final
(diluted) buffers should not be adjusted using a pH electrode, if
possible only adjust pH of stocks. If it cannot be avoided, either pH
292
Simone Kunzelmann
kinetics of enzyme reactions in real time, in steady state as well as
pre-steady state experiments.
Steady state kinetic assays allow determination of k cat , K m , and
the kinetic efficiency k cat /K m , as well as the study of activation and
inhibition of an enzyme. Biosensors with very fast response times,
like PBP, also provide the time resolution needed for pre-steady
state, kinetic experiments using flash photolysis, or stopped-flow
techniques, which can give a more detailed insight into the mechanism of the enzyme reaction.
In this chapter, I describe how to use phosphate-binding protein to measure the kinetics of ATPase and GTPase reactions.
Protocols are given for determining steady state kinetic parameters,
K m and k cat , in a plate reader format in the absence and presence of
an activator. In addition, I describe examples of direct measurement
of P i release under pre-steady state conditions using stopped-flow
and illustrate what information about the enzyme mechanism can
be deduced from this type of data.
2 Materials
2.1 Phosphate
Biosensor
The coumarin version of the biosensor, MDCC-PBP (see Note 1),
is sold as ready-to-use phosphate sensor by Thermo Fisher Scientific (PV4406/7), but the biosensor used for the experiments in
this chapter was purified and labeled in the laboratory, which is a
straight forward procedure. Rho-PBP is not currently available
commercially. The latest expression vectors for both biosensors
are available from Addgene (http://www.addgene.org/): plasmids
pET22b_PstS_1 for MDCC-PBP and pET22b_PstS_2 for
Rho-PBP [9]. Detailed protocols for purification, labeling, and
characterization of the biosensors can also be found on the
Addgene webpage. Note that the expression system has changed
to IPTG induction [9] since the original publications [1, 5, 6].
2.2 Reaction Buffer
The most critical factor for successful assays of P i is to avoid contamination with P i , which is a very widespread impurity (see Note
2).
All buffer components should be analytical grade. Water used
for buffer preparation and rinsing should be used directly from the
distillation apparatus or Milli-Q system without storage to avoid
contamination. We have worked successfully with double-distilled
water, Milli-Q, and commercial water (HPLC grade). Disposable
plasticware is used where possible and everything is rinsed with
P i -free water before use. If glassware cannot be avoided, it is soaked
in 25% HCl overnight and rinsed with water. The pH of final
(diluted) buffers should not be adjusted using a pH electrode, if
possible only adjust pH of stocks. If it cannot be avoided, either pH
292
Simone Kunzelmann