Student guide
Concluding module
Toothpickase +
Developed by TU Delft and edited by bioinformaticsaschool.eu (part of NBIC)
Text
Dr. T. Klop and Hienke Sminia
Illustrations
Sebastiaan Donders (via www.allesoverDNA.nl)
Bioinformaticaindeklas.eu
All the included material is protected by the Creative Commons
Naamsvermelding-Niet-commercieel-Gelijk delen 3.0 Nederland
license (http://creativecommons.org/licenses/by-nc-sa/3.0/nl/).
CC BY-NC-SA 2009 – Netherlands Bioinformatics Centre
For any questions or comments, please contact the Travelling DNA Labs ([email protected]).
Student Guide Module Toothpickase +
Student Guide
Introduction
An enzyme is a protein which speeds up chemical reactions or allows for them to happen altogether.
Enzymes are essential for the body to function properly. For example, the enzyme ‘amylase’ in human
saliva speeds up the processing of potato starch into small sugar molecules. In this reaction, the
enzyme isn’t broken down: it stays intact. Therefore, when it finishes reacting, this enzyme is
immediately available for the next reaction.
In the DNA Lab ‘Bioinformatics: life in the computer’, you have looked at snake venom in detail. This
venom is also an enzyme: it breaks down collagen.
In the next experiments, you will act as the enzyme ‘toothpickase’, which breaks down toothpicks (the
substrate, or ligand) in certain time intervals. You will calculate how many toothpicks are being broken
down each second. Doing so, you can determine the reaction speed of the toothpickase enzyme.
Materials
 ± 150 toothpicks per team
 Dish/bowl
 Stopwatch
 Paperclips, 10 per team
 Pen
Method
Using only your hands and the toothpicks, you will reconstruct ho wan enzyme
works. The enzyme ‘toothpickase’ is represented by your thumb and index
Active
finger of one hand in a specific shape. The active site (see figure) is the part
site
between thumb and index finger, in which the toothpick (‘substrate’) will sit. This
way, the toothpick can be broken into two pieces (product).
Enzyme
The speed of this reaction can be measured by counting the amount of broken
toothpicks (product). There are many factors that can influence the enzyme’s
A schematic
reaction speed.
representation of an
 Find the right shape for your toothpickase.
enzyme
 Break a toothpick above a bowl.
 Try another shape of toothpickase.
 Try a couple of shapes to find out which works best for you..
Substrate
Given is a stack of toothpicks under ideal conditions; then still, it will take some time for the enzyme to
break all the toothpicks. This is the V-max of the enzyme (the speed at which the product is
generated). If the whole classroom would be full of toothpicks, this speed would not change.
1. If the toothpick would be in the other end of the classroom, would it take more time to break a
toothpick? Explain your answer using the term substrate concentration.
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Student Guide Module Toothpickase +
2. Would it take more time to break a stack of toothpicks if there were also paperclips in the
stack? Explain your answer using the term competitive inhibitor.
3. What would happen with the time needed to break down all the toothpicks if 2 people would
do this at the same time? Explain your answer using the term enzyme concentration.
Do the next experiments in groups of at least 4 people
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Student Guide Module Toothpickase +
Experiment 1: Reaction speed of toothpickase
 Put approximately 60 tootpicks in the bowl.
 Break as many toothpicks as possible between thumb and index finger without looking.
 Warning! Throw the broken halves back in the bowl (product and substrate are mixed in a
reaction), but a toothpick may only be broken once (the enzyme has reacted with the
substrate before).
 Write down the amount of broken toothpicks in intervals of 10 seconds in table 1.
Table 1
Time (seconds)
Amount of broken
toothpicks (N)
Average speed
0
10
20
30
40
50
60
70
80
90
Total
1. Does the amount of broken toothpicks change over time? Explain your answer.
2. How many toothpicks are broken in total during the 90 seconds? Compare your answers to that
of classmates. Why have others broken more or less toothpicks?
3. Calculate the average reaction speed between time intervals, using this formula (slope): N 2 -N 1 /
T 2 -T 1 and fill out the last column of table 1.
If you do this for 180 seconds, the reaction speed in the interval 120 – 180 seconds will probably be
0.
4. Explain that the amount of available substrate has consequences for the reaction speed of the
enzyme. In your answer, use amylase (from saliva) as an example.
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Student Guide Module Toothpickase +
5.
What is the substrate in the case of the snake venom?
Experiment 2: Adding an inhibitor




Put approximately 60 new toothpicks in the bowl.
Mix 10 paperclips through the toothpicks.
Again, break as many toothpicks as possible between thumb and index finger without looking.
Write down the amount of broken toothpicks in intervals of 10 seconds in table 2.
Table 2
Time (seconds)
Amount of broken
toothpicks (N)
0
10
20
30
40
50
60
70
80
90
1. Which effect does adding a ‘wrong substrate’ to the amount of toothpicks broken by
toothpickase? Explain your answer.
In medicine development, these ‘wrong substrates’ are used a lot. The medicines usually have a
higher affinity (more efficient binding) to the enzyme than the normal substrate.
2. Explain that the antivenom from the practicum works in a similar fashion.
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Student Guide Module Toothpickase +
Conclusion
In the drawing space below, plot a graph using the data from table 1 (experiment 1) by placing dots
and connecting them. Also plot the graph of the data of table 2 (experiment 2) in this same drawing
area. Use different colors for each graph. Add a suitable caption in the box below. Finally, answer the
following questions.
# broken toothpicks
Graph: Efficiency toothpickase
Time (seconds)
Graph 1: (caption)
1. Give a general description of an enzyme. What was the enzyme in this experiment?
2. Give a general description of a substrate. What was the substrate in this experiment?
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Student Guide Module Toothpickase +
3. Describe how adding another substrate affects the efficiency of the enzyme. Use the graph to
explain your answer.
4. What happens to the reaction speed of an enzyme if the substrate concentration decreases?
5. Describe how temperature affects the action of enzyme. What causes a more definitive change in
the enzyme: heating or cooling?
6. How would you investigate this heat effect, using a similar set-up as in this toothpickase
experiment?
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Student Guide Module Toothpickase +
Supplements
Examples of applications of enzymes in our daily lives.
Application
Nutrition
Bakery
Enzyme and function
Paper
Lipase breaks down lipids in resin, which interferes with paper production (makes
stains and holes in the paper)
Laccase breaks down lignine in wood, because this causes an unwanted brown
color
Lipase breaks down remaining tissue attached to the leather
Protease breaks down proteins, which makes the leather easier to depilate, and
increases its flexibility
Amylase homogenous crumb structure and big bread volume
Maltogen longer preservability of bread
Alpha-amylase the first enzyme on the market to be produced by genetically
modified organisms (GMOs)
Xylanase dough easier to handle and better crumb
Dairy
Chymosine curdles milk to make cheese
Protease breaks down allergen proteins
Lipase improves ripening of cheese
Brewery
Amylase breaks down starch into fermentable sugars
Beta-glucanase prevents 'chill haze' in beer (becoming cloudy when cooled down)
Fruit juices
Pectinase / Amylase improves juice yield and clearness
Wine
Pectinase maintains colour and clearness
Livestock nourishment
Fytase makes livestock nourishment phosphor-free, making it easier to absorb for
animals
Non-nutrition
Textile
Amylase breaks down starch in the wax layer that is applied to textile fibers to
protect them during weaving
Cellulase alternative for stonewashing
Laundry
Cellulase breaks down cellulose into smaller components that can be easily
products
washed off
Lipase breaks down lipids, the most important component of oils and grease
Protease breaks down proteins into smaller components that can be easily washed
off
Leather
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