Question

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 to an allosteric site leading to a reduction in

activity.

The binding of ATP can be measured using a fluorescently labelled derivative of ATP – TNP-ATP. The data

presented below represents the determination of the binding of ATP 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 was carried 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. Only plot the data points – do

not join data points with lines. Ensure that each dataset is distinct from the others (use

alternate colours and/or shapes for the data points).

b) Fit an equation to each dataset separately. Do not show these on your plots. For each

experiment give the fitted equation and the value of Kd. Show these in a table. Ensure you

include the units of this value. Show your working for the Experiment 1 data.

c) Determine the mean and standard deviation of the data for each 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 the calculated Kd for the averaged

data. Add a trendline defined by this equation to your graph.

d) With reference the data you plotted and Kd values you determined, explain the positive

and negative aspects of using a double reciprocal plot.

Answer 1

Values for inverese plot

1/[TNP-ATP]	1/ Exp 1	1/Exp 2	1/Exp 3
0.016667	12.82051	12.34568	11.76470588
0.008333	7.633588	7.194245	7.142857143
0.004167	4.166667	4.149378	4
0.002083	3.076923	2.985075	3.134796238
0.001042	2.403846	2.262443	2.444987775

to determine the Kd or Km, double reciprocal plot used. This follow the straight line equation where a= slope and Yo = intersept or Vmax.  

In this equation slope= Kd/Vmax= slope for each equation is given and Vmax or incept is alos given now calculate the kd value

Here Y= 1/Vmax

Vmax= 1/Y

For first graph

676= kd/ (1/1.65)= 409 uM

Kd2=648* 0.628 = 407 uM

Kd3=603*0.55=335 uM

1/[TNP-ATP]	mean	SD
0.0167	12.3103	0.5288
8.33E-03	7.3236	0.2697
4.17E-03	4.1053	0.0916
2.08E-03	3.0656	0.0755
1.04E-03	2.3704	0.0958

Inverse plot increase the errors.