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BC1B Ligand-Binding Assignment 2024
Instructions
This assignment builds on your experience with Graphpad Prism in the Ligand Binding
workshop and makes use of it to analyse a different kind of binding/activity data. This
is an individual assignment – your answers should be all your own work and should
be written in your own words. Your assignment will be submitted to Turnitin for the
detection of plagiarism or collusion.
You should use MS Word (or similar) to present the graphs exported or copied from
Prism and written answers to this assignment. You can create other figures either in
a drawing app (such as Powerpoint), and pasting it in to your Word file, or by drawing
by hand, taking a picture and inserting the picture into your Word document.
Please do not paste the questions into your Word document. It should contain your
answers to the questions only.
Please write your seven digit University Number at the top of your Word document.
You should not write your name – marking is done anonymously.
Please save your Word file with the words “BC1B Ligand Binding” followed by your
University Number e.g.
“BC1B Ligand Binding 2345678.docx”
Please submit your completed Word file by following the instructions on the
“Assessment, submission and feedback” page of the BC1B Blackboard site.
The deadline for submission of your Word file is 2pm on Wednesday 28th February
2024 (Week 18).
Should you encounter a problem with your submission, please email bioc_xfffe_teaching@bristol.ac.uk for guidance.
Assessment Task
The glycolytic enzyme phosphofructokinase (PFK) was incubated with a constant
concentration of ATP and increasing concentrations of fructose 6-phosphate (F-6-P).
The following data were obtained (where %Vmax is the enzyme activity expressed as
a percentage of the maximal activity under fully saturated conditions).
[F-6-P] (mM) %Vmax
0.0 0.0
0.2 2.0
0.4 12.5
0.6 30.8
0.8 50.0
1.0 65.1
1.2 75.7
1.4 82.7
1.6 87.4
1.8 90.6

Q1: Comment on the shape of the curve described by these data. (5 marks)
In Graphpad Prism, start a new project as you did in the workshop and enter the above
data into the data table. Check that it has also plotted a graph.
You need to fit your data to the Hill equation using Prism:
=
.[ ]

+ [ ]

(where n is the Hill constant and K is the substrate concentration at which the
enzyme has half-maximal activity)
In Prism, starting with your graph selected: select Analyze > XY Analyses > Non-linear
regression (curve fit) and then press OK. In the pop-up menu under the Fit tab, select
“New” on the right hand side and then “Create New Equation”. In the next pop-up
dialogue box give your equation the name “Hill equation” and in the “Definition” box
enter the following equation:
Y=Vmax*X^n/(K^n+X^n)
Under the “Rules for Initial Values” tab enter the following:
Parameter Name Initial Value Rule
Vmax 1 *YMAX
n 1 (leave this one)
Khalf 1 *(Value of X at YMID)
At the bottom under the heading “Default Range”
Select: Start graphing the curve at “X=0”
If you press OK then Prism should fit your data to the Hill equation you just entered.
Q2: (a) Paste a copy of your graph with the fitted data into your answer sheet.
(5 marks)
(b) What are the fitted parameters: Vmax, n and K (10 marks)
Q3: With the help of a cartoon sketch (or using Powerpoint) describe the
Monod-Wyman-Changeux (MWC) model for the enzyme PFK. (15 marks)
You can perform a linear transform of these data to the linear form of the Hill equation:

( )
= . log[ ]
As with the Lineweaver-Burk in the workshop (see Exercise 5), Prism can do this
transform automatically. However, instead of choosing the “Lineweaver-Burk (double
reciprocal)” choose “Hill”, enter Vmax as 100 and change “X values are entered as” to
“concentration”. Check your new graph to make sure that the data look linear.
Ensuring the new graph of the linear transformed data is open, fit these data to a linear
regression. The gradient is n and the X-intercept is log.K.
Q4: (a) Paste your graph with linear fit into your answer document. (10 marks)
(b) What are the fitted values of n and K (5 marks)
(c) Comment on the values of n and K compared to Q2. (5 marks)
The experiment was repeated in the presence of 0.1mM AMP with the following
results:
[F-6-P] (mM) %Vmax
0.00 0.0
0.025 15.0
0.05 27.6
0.10 45.0
0.15 56.1
0.20 63.7
0.30 73.2
0.40 79.0
0.50 82.8
Using a new Data Table, plot these data in Prism and inspect the new graph.
Q5: What do you notice about these data compared to the previous set in the
absence of AMP (5 marks)
As you did for the previous data, fit these data to the Hill equation you just created.
Q6: (a) Paste your graph with fitted line into your answer document. (10 marks)
(b) What are the fitted parameters: Vmax, n and K (10 marks)
Q7: With reference to the MWC model figure you drew for Q3, explain the
action of AMP on PFK. (10 marks)
Q8: Briefly outline why this is important for the regulation of glycolysis. (10
marks)