3 Methods
3.1 Steady State
Kinetic Assay
to Determine K m
and k cat
In steady state kinetics experiments, one measures the initial rate at
which substrate (S) is turned over by a small amount of enzyme
([E] 0 ( [S]), most commonly at different concentrations of substrate to determine K m and k cat . These assays are also used to study
the mechanism of activation or inhibition by other molecules and
the apparent affinities of activators and inhibitors.
Here, it is described how to use phosphate-binding protein to
measure K m and k cat of a P i -producing enzyme using a fluorescence
plate reader. I describe general considerations for the experimental
design and procedure and present a detailed example of characterizing the ATPase activity of Chd1, an ATP-dependent chromatin
remodeler, and its activation by DNA. Formation of P i in real time
is measured using MDCC-PBP. K m and k cat for ATP hydrolysis by
Chd1 are determined in the absence and presence of
activating DNA.
3.1.1 General Principles
To measure initial rates, enzyme and substrate are mixed in the
presence of MDCC-PBP and the fluorescence signal is monitored
over time (see Fig. 2a).
A linear fit to the data within the steady state phase gives the
rate in fluorescence units per time interval. Using a calibration with
a P i standard (see Fig. 3), the rate of fluorescence change is converted into the rate of P i produced.
To determine K m and k cat for an enzyme–substrate reaction,
initial rates are measured using a range of substrate concentrations.
The dependence of the initial rate, V, on substrate concentration
[S] is described by the Michaelis–Menten equation, which is in its
original form:
V ¼
V max Á S
½
K m þ S
½
with V max ¼ k cat Á E
½ 0
ð1Þ
Often it is useful to calculate and plot the specific rate ν, which
is the initial rate normalized to the enzyme concentration ν ¼ V/
[E] 0 (see Fig. 2b). The specific rate ν is given by
ν ¼
k cat Á S
½
K m þ S
½
ð2Þ
Presenting the data on a plot of specific rate, ν, versus substrate
concentration, [S], according to Eq. (2), enables the direct visualization of k cat (ν at saturating concentration), as well as the comparison of multiple datasets, which have been measured at different
enzyme concentrations. It is common practice to vary enzyme
concentration as appropriate, to accurately measure reactions with
Phosphate Biosensor Assays
295
3.1 Steady State
Kinetic Assay
to Determine K m
and k cat
In steady state kinetics experiments, one measures the initial rate at
which substrate (S) is turned over by a small amount of enzyme
([E] 0 ( [S]), most commonly at different concentrations of substrate to determine K m and k cat . These assays are also used to study
the mechanism of activation or inhibition by other molecules and
the apparent affinities of activators and inhibitors.
Here, it is described how to use phosphate-binding protein to
measure K m and k cat of a P i -producing enzyme using a fluorescence
plate reader. I describe general considerations for the experimental
design and procedure and present a detailed example of characterizing the ATPase activity of Chd1, an ATP-dependent chromatin
remodeler, and its activation by DNA. Formation of P i in real time
is measured using MDCC-PBP. K m and k cat for ATP hydrolysis by
Chd1 are determined in the absence and presence of
activating DNA.
3.1.1 General Principles
To measure initial rates, enzyme and substrate are mixed in the
presence of MDCC-PBP and the fluorescence signal is monitored
over time (see Fig. 2a).
A linear fit to the data within the steady state phase gives the
rate in fluorescence units per time interval. Using a calibration with
a P i standard (see Fig. 3), the rate of fluorescence change is converted into the rate of P i produced.
To determine K m and k cat for an enzyme–substrate reaction,
initial rates are measured using a range of substrate concentrations.
The dependence of the initial rate, V, on substrate concentration
[S] is described by the Michaelis–Menten equation, which is in its
original form:
V ¼
V max Á S
½
K m þ S
½
with V max ¼ k cat Á E
½ 0
ð1Þ
Often it is useful to calculate and plot the specific rate ν, which
is the initial rate normalized to the enzyme concentration ν ¼ V/
[E] 0 (see Fig. 2b). The specific rate ν is given by
ν ¼
k cat Á S
½
K m þ S
½
ð2Þ
Presenting the data on a plot of specific rate, ν, versus substrate
concentration, [S], according to Eq. (2), enables the direct visualization of k cat (ν at saturating concentration), as well as the comparison of multiple datasets, which have been measured at different
enzyme concentrations. It is common practice to vary enzyme
concentration as appropriate, to accurately measure reactions with
Phosphate Biosensor Assays
295