Code
try:
import fysisk_biokemi
print("Already installed")
except ImportError:
%pip install -q "fysisk_biokemi[colab] @ git+https://github.com/au-mbg/fysisk-biokemi.git"
Use the widget created by the cell above to answer the following the questions.
Setting \(V_\mathrm{max} = 400\) and \([E]_\mathrm{tot} = 0.0005\), change \(K_M\) from 0.5 to 5 to 10 to 50. Describe the change in curve appearance and explain it using the Michaelis-Menten (MM) equation.
What is the biological meaning of \(K_M\)?
Now set \([E]_\mathrm{tot} = 0.0005\), and \(K_M = 5\), change \(V_\mathrm{max}\) from 40 to 100 to 900. Do you observe any changes? Explain your answer using the widget and the MM equation
Now set \(K_M = 5\) and \(V_\mathrm{max} = 100\), change \([E]_\mathrm{tot}\) from 0.0005 to 0.05. Do you observe any change in curve appearance, in kcat or in kcat/KM? Explain your answer
Consider a reaction catalyzed by an enzyme obeying the Michaelis-Menten kinetics model.
\[ E + S \underset{k_{-1}}{\stackrel{k_1}{\rightleftharpoons}} ES \stackrel{k_2}{\rightarrow} E + P \]
\[ V_0 = V_{\mathrm{max}} \frac{[S]}{[S] + K_M} \]
Explain what is meant by enzyme saturation.
Calculate the level of enzyme saturation, \(f_{ES}\), at substrate concentrations of
The Michaelis-Menten constant KM is defined as:
\[ K_M = \frac{k_{-1}+ k_2}{k_1} \]
In a specific reaction, the following rate constants were determined:
Calculate the value of \(K_M\). Does \(K_M\) approximate the dissociation constant for the \(ES\) complex in this case?
Assume that a solution of an enzyme at a concentration of \(1 \cdot 10^{-7} \ \mathrm{M}\) with a substrate concentration of \([S]=100\cdot K_M\) has \(V_0=10^{-4} \ \mathrm{M} \cdot \mathrm{min}^{-1}\)
Given this information and with your answers to (b) and (c) in mind, calculate the constant \(k_2\). What is this constant also called?
The values of \(V_\mathrm{max}\) and \(K_M\) have historically been determined from a Lineweaver-Burk plot.
How does the x- and y-intercepts in a Lineweaver-Burk plot relate to \(V_\mathrm{max}\) and \(K_M\)?
An enzyme obeying the Michaelis-Menten kinetics model was tested for substrate conversion in the absence and presence of an inhibitor, called inhibitor1 at a concentration of \([\textrm{I}] = 2.5 \cdot 10^{-3} \ \textrm{M}\). The data set is contained in the file enzyme-inhib-i.xlsx
. Using this data a researcher wanted to determine the type of inhibition.
Start by loading the dataset
Run the next cell after uploading the file
Convert the concentrations of substrate and the initial velocities to units given in M and \(\mathrm{M}\cdot \mathrm{s}^{-1}\), respectively.
Plot the initial velocities of both experiments as a function of substrate concentration in one plot. Estimate \(k_\mathrm{cat}\) and \(V_\mathrm{max}\) in the presence and absence of inhibitor from the plot.
The researcher wanted to determine \(V_\mathrm{max}\) and \(K_M\) values for both experiments in order to correctly conclude on the type of inhibitor.
Determine \(V_\mathrm{max}\) and \(K_M\) by fitting.
Start writing the function to fit with
And use the curve_fit
-function to find the parameters
What type of inhibitor is inhibitor1?
Calculate \(K_i\).
Two enzyme inhibitors were identified as part of a drug discovery program. To characterize the mechanism of action, the reaction kinetics were analysed for the enzyme alone and in the presence of 5 \(\mu\)M of each of the inhibitors as a function of substrate concentration resulting in the data in the file enzyme-inhib-ii.xlsx
Load the dataset
Run the next cell after uploading the file
Convert the measurements to SI units and plot the reaction rate against time for all three reactions.
Start by converting the units
And then plot
fig, ax = plt.subplots()
col_names = ['enz_(M/s)', 'inhibitor2_(M/s)', 'inhibitor3_(M/s)']
for col_name in col_names:
ax.plot(..., ..., 'o', label=col_name) # Replace ... with your code here.
ax.legend()
ax.set_xlabel('Substrate concentration [M]', fontsize=14)
ax.set_ylabel('$V_0$', fontsize=14)
plt.show()
Based on the appearance of these plots:
Based on your answer to in (a) what do you expect for the parameters \(K_M\) and \(V_\mathrm{max}\)?
As always, we need the function we’re fitting with
Given we’re fitting on multiple datasets we will make our lives a bit easier by making a little function
In the fits above you will likely see that all the fitted parameters change between the different data sets – some by a little and some by a lot. How big does a change have to be to be considered significant?
Determine the \(K_i\) for each of the two inhibitors.
When (or if depending on your outlook in life) you are bit by a cobra, the snake protein CVF interacts with the human protein FB to form a proteolytically active enzyme in the blood called CVF:FB. CVF:FB enzyme is able to cleave another human protein known as C5 into C5b and C5a (C5a is the protein you visually inspected in your first PyMOL session). With C5 as a substrate, CVF:FB obeys the Michaelis-Menten kinetics model, whereas CVF alone does not show any catalytic activity towards C5.
Now consider two different CVF proteins, CVF1 and CVF2 from two different cobra species. Upon characterization it was found that CVF1:FB has a \(K_M = 0.036 \ \mu\text{M}\) and a \(k_{cat} = 0.43 \ \mathrm{min}^{-1}\), whereas CVF2:FB has a \(K_M = 14 \mu\mathrm{M}\) and a \(k_{cat} = 0.73 \ \mathrm{min}^{-1}\). The concentration of C5 in blood is \(0.37 \ \mu\mathrm{M}\).
Convert the given \(K_M\) values and the C5 concentration to SI-units given in \(\mathrm{M}\)
Assuming that [CVF1:FB] \(=\) [CVF2:FB] \(= 0.1\cdot 10^{-6} \ \mathrm{M}\), calculate \(V_\mathrm{max}\) of both enzymes.
Calculate the fraction of enzyme:substrate complexes (CVFx:FB:C5) in blood.
Calculate the initial velocities of C5 cleavage in blood by the two enzymes.
Which of the two enzymes, CVF1:FB or CVF2:FB, cleaves the most C5 molecules in blood?
The enzyme aspartate transcarbamoylase (ATCase) catalyzes the first reaction in the biosynthesis of pyrimidines such as CTP as shown in the reaction below:
ATCase does not obey the Michaelis-Menten kinetics model but instead shows the behaviour recorded in the enzyme-behav-atcase.csv
dataset.
The dataset consists of three columns; the aspartate concentration in mM and the rate of formation of N-carbamyolaspartate with and without the presence of CTP.
Load the dataset with the widget below
Run the next cell after uploading the file
Describe the kinetic profile of ATCase and explain what it tells us about the way ATCase works. (You may find inspiration in the material previously covered on protein-ligand interactions)
What does the figure tell us about the quaternary structure of ATCase?
Which other pyrimidine based trinucleotide besides CTP is used to synthesize DNA?
Qualitatively describe the effect of CTP on the rate of N-carbamyolaspartate formation
In fact many enzymes are regulated by certain end products in a fashion similar to the CTP effect on ATCase. Can you explain why this might be a physiological advantage?
---
title: Week 48
engine: jupyter
categories: ['regression', 'calculation', 'data', 'plotting']
format-links:
- text: "Open in Google Colab"
href: "https://colab.research.google.com/github/au-mbg/fysisk-biokemi/blob/built-notebooks/built_notebooks/student/week_48.ipynb"
icon: box-arrow-up-right
---
{{< include tidbits/_install_import.qmd >}}
---
{{< include exercises/quali-descr-michae-kineti.qmd >}}
---
{{< include exercises/michaelis-menten.qmd >}}
---
{{< include exercises/enzyme-inhib-i.qmd >}}
---
{{< include exercises/enzyme-inhib-ii.qmd >}}
---
{{< include exercises/snake-dependent-enzyme.qmd >}}
---
{{< include exercises/enzyme-behav-atcase.qmd >}}