The PFK1 enzyme is regulated by a number of factors in the cell,including the level of ATP. ATP is not only
involved in the reaction the enzyme catalyses but also binds toan allosteric site leading to a reduction in
activity.
The binding of ATP can be measured using a fluorescentlylabelled derivative of ATP – TNP-ATP. The data
presented below represents the determination of the binding ofATP to PFK. The change in fluorescence (?
fluor.) represents the concentration of PFK1 with TNP-ATP bound.This value was determined at 5
different concentrations of TNP-ATP, and the experiment wascarried out in triplicate.
| ? fluor. (arbitrary units) |
[TNP-ATP] (µM)   | Experiment 1 | Experiment 2 | Experiment 3 |
60 | 0.078 | 0.081 | 0.085 |
120 | 0.131 | 0.139 | 0.140 |
240 | 0.240 | 0.241 | 0.250 |
480 | 0.325 | 0.335 | 0.319 |
960 | 0.416 | 0.442 | 0.409 |
a) Create a double reciprocal plot of this data (1/[TNP-ATP] vs.1/ ? fluor.). Plot the data from
each experiment as a separate dataset on the same graph. Onlyplot the data points – do
not join data points with lines. Ensure that each dataset isdistinct from the others (use
alternate colours and/or shapes for the data points).
b) Fit an equation to each dataset separately. Do not show theseon your plots. For each
experiment give the fitted equation and the value of Kd. Showthese in a table. Ensure you
include the units of this value. Show your working for theExperiment 1 data.
c) Determine the mean and standard deviation of the data foreach concentration of TNPATP.
Show these in a table. Transform this data as you have done in(a). Fit an equation to
the transformed data. Give the fitted equation and thecalculated Kd for the averaged
data. Add a trendline defined by this equation to yourgraph.
d) With reference the data you plotted and Kd values youdetermined, explain the positive
and negative aspects of using a double reciprocal plot.
