Name:______
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 secondsduring 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?