Download Enzyme Kinetics Lab

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The Enzyme Kinetics Lab
Without enzymes, the dynamic steady state called “you” would quickly cease to exist.
Reactions simply would not proceed fast enough for the body to process food, send signals
among brain cells, make muscles contract, and do everything else to stay alive.
Enzymes are a class of proteins that greatly speed up (catalyze) reactions between specific
substances, usually at their functional groups. The substances that each type of enzyme acts
upon are called its substrates. Enzymes have four common features:
1. They don’t make anything happen that couldn’t happen on its own.
2. They are not permanently altered or used up.
3. The same enzyme works for both the forward and reverse reaction.
4. Each enzyme is highly selective about its substrate.
The rate of reaction of an enzyme is affected by many factors such as temperature, and pH.
But as you’ll see in this lab activity, the reaction rate of an enzyme can also be affected by the
concentration of substrate, competitive inhibition, and noncompetitive inhibition.
Materials:
100 colored coins (pennies)
10 uncolored coins (nickels)
Stopwatch
Adhesive tape
Cloth blindfold
Activity 1: Simulating enzyme activity and substrate concentration
1. Work in groups of four.
2. Assign the following jobs: Enzyme (coin collector), Timer, Data recorder, and
experimental setup.
3. Blindfold your enzyme volunteer.
4. Clean your lab tables and evenly place 10 colored coins (pennies) on the table. This
represents the substrate concentration. In this activity you are going to test to see what
happens to an enzymes rate of reaction (pennies collected) versus the amount of substrate
(pennies on the table).
5. Now the timer needs to time the “enzyme” for exactly 30 seconds during which the
blindfolded student collects coins with only one hand and placed them in a cup held in their
second hand.
6. Multiply the number of coins collected in 30 seconds by two and then record the number of
substrate (pennies) that your enzyme was able to react with in data table 1 below.
7. Repeat using the same enzyme using 20, 30, 40, 50, 50, 70, 80, and 90 colored coins. Be
sure to evenly place them on the table.
8. Create a data table containing the independent and dependent variables.
Analysis:
1. How does substrate concentration (number of pennies) affect the enzyme activity
(number collected)?
2. Around 50-70 coins the number of substrate molecules that the enzyme is able to
react with (collect) seems to level off. Why? What is happening?
Activity 2: Competitive inhibition
In competitive inhibition, the inhibitor resembles the substrate and carries out reversible binding,
but the reaction actually does not once the inhibitor is bound. While the inhibitor occupies the
active site, it prevents binding by the substrate until the release of the inhibitor from the enzyme
takes place.
1. In order to visualize the effect of competitive inhibitors on the system, repeat the simulation
above but this time add 10 uncolored coins (nickels) among the different numbers of colored
coins. The nickel simulates the competitive inhibitor.
2.
Analysis:
3. How do competitive inhibitors (nickels) effect the reactivity of your enzyme with the
correct substrate (pennies collected)?
4. At what point does your enzyme reach saturation? (At what point does the number of
pennies collect seem to level off?) Does this saturation point occur at the some point as
in activity one?
Activity 3: Noncompetitive Inhibition
In noncompetitive inhibition, the inhibitor binds to the enzyme and causes changes in the
enzyme configuration and results in a partial inactivation of the enzyme. Basically, the inhibitor
makes it very difficult for the enzyme to function properly.
1. To simulate a noncompetitive inhibitor, tape together the middle three fingers of your
enzyme.
2. Repeat the activity as in part 1 without the competitive inhibitors (nickels) from part 2.
3. Record you data in the table below. Record the number of colored coins that your enzyme is
able to collect for each 30 time period by creating a data table containing the independent and
dependent variables.
Analysis:
5. What happens to the enzymes ability to react with the substrate (collect pennies)
when a noncompetitive inhibitor is bound to it?
6. Which inhibitor (competitive or noncompetitive) seems to affect the reactivity of the
enzyme the most? Try and explain why this happens?