BIOSC-116 Lecture Notes - Lecture 24: Trypsin, Pepsin, Cytochrome C Oxidase
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Enzyme Kinetics Simulation Experiment
Adapted from Junker, M. J. Chem. Educ. 2010, 87, 294-295.
Objective:
To simulate the working of enzymes in the catalysis of biochemical reactions.
Materials:
Bolts (15) and nuts to match, purchased at a hardware store or found in your basement. If you donât have any nuts and bolts, use anything made of two parts that can be taken apart. For example, red and blue paper clips, large and small paper clips, baby food jars and lids. Use your imagination. In addition, you will need something to glue the two pieces together. You will also need a device to time yourself, a watch with a second hand or a stop watch.
Background Information and Theory:
Read pp. 655-672 in Seager and Slabaugh. Most of the metabolic reactions in Chapters 23 and 24 are catalyzed by enzymes. Enzymes work by lowering the activation energy for biochemical reactions. The first step is for the substrate (S), the reactant to react with the enzyme (E) to form the Enzyme-Substrate Complex (ES):
E + S ES. The second step is for the enzyme to convert the substrate into the product(s). In this case there will be two products, P1 and P2: ES P1 + P2. In this simulation the substrate will be the nut and bolt screwed together. You will be the enzyme. As the enzyme, it will be your job to find the substrate and unscrew the nut from the bolt. The nut will be P1 and the bolt will be P2. The simulation will simulate the case where the substrate concentration is less than saturation. (Figure 20.6, p. 663), below maximum velocity). It will also simulate saturating the substrate concentration. (Figure 20.6, p. 663, at maximum velocity). Competitive Inhibition, (p. 668) and non-competitive inhibition (p. 669) will be simulated as well.
Procedure:
Enzyme Kinetics When Substrate Concentration is Less Than Saturation
Screw the 15 nuts on to the 15 bolts. Scatter the bolts randomly around the room you are in. Start the timer and gather and unscrew as many nuts as possible in 30 seconds. Record the number of bolts unscrewed on the table/report sheet in Doc Sharing. (I have also placed the table following the write-up on the main page, here. Divide the number of bolts unscrewed by the time (0.5 minutes) to get the rate of the reaction.
Enzyme Kinetics When Substrate is at Saturation
Reattach the 15 nuts to the bolts. Put all the nut/bolt combinations in front of you on the table. Start the timer and unscrew as many nuts as possible in 30 seconds. Record the number of bolts unscrewed on the report sheet. Divide the number of bolts unscrewed by the time (0.5 minutes) to get the reaction rate.
Enzyme Kinetics With Competitive Inhibition
Reattach the nuts to the bolts. Glue 10 of the nuts to the bolts, so they canât be removed. Place the nut/bolt combinations in a pile. Unscrew as many of the nuts as possible in 30 seconds. Count the number of unscrewed nuts. Donât try to muscle the glued ones. They canât be removed. Count the number of nuts unscrewed. Record this number in the table. Also record the reaction rate by dividing by the time (0.5 minutes)
Enzyme Kinetics With Non-Competitive Inhibition
Reattach the nuts to the bolts. Place the nut/bolt combinations in front of you on the table. Have someone tie your hands behind your back, or mentally tie your hands behind your back. Start the timer and see how many nuts you can remove from the bolts. Record the numbers of nuts removed, and divide by time (0.5 minutes) to get the reaction rate.
Fill out table below.
Condition | Number of Bolts Unscrewed | Time (minutes) | Reaction rate (bolts per minute) | Comment |
Scattered bolts | Non-saturated substrate | |||
Pile of Bolts | Saturated Substrate | |||
Some Bolts Glued | Competitive Inhibiter | |||
Arms tied | Non-Competitive Inhibitor |
Answer these questions:
How well do you think this simulation represents enzyme kinetics? Did it help you understand kinetics better? Can you think of anything that could be added to improve the simulation?
Enzymes Lab Report
Biology 1408
- Temperature and Enzyme Activity
- Based on Lab, Section III, Exercise 3 record the relative colorintensity at five-minute intervals to determine the temperaturerange for catechol oxidase.
Time (min) | Tube 1 4oC | Tube 2 23oC | Tube 3 40oC | Tube 4 60oC | Tube 5 80oC | Tube 6 100oC |
0 | ||||||
5 | ||||||
10 |
- Construct a bar graph of the data for the ten-minute readings.Label axes and provide units. Sign, date and prepare an image ofyour graph and include it with this lab report.
- What is the optimal temperature range for catechol oxidaseactivity?
- pH and Enzyme Activity
- Within what pH range will catechol oxidase catalyze catecholand oxygen to form benzoquinone?
4-12
- Define pH.
- What is the role of catechol in the experiment?
- Which test tube contains the most acidic buffer solution?
- Cofactors and Enzyme Activity
- What is a cofactor?
- What cofactor is necessary for catechol oxidase activity?
Summary Questions
- What was the role of catechol oxidase in the experiments?
- What was the purpose of developing a color intensityscale?
- Identify three factors that influence the rate of enzymaticreactions.
- Examine the data in Exercise 3 and suggest a hypothesisexplaining why people with a fever feel tired and listless.
With high fever, enzymestructure changes and no longer act as a catalyst
- People who have been suddenly submerged in cold water andremained for long periods of time have survived. Examine the datain Exercise 3 and explain how this is possible.
Low temperature ranges willinhabit the reaction of the speed of chemical reaction incells
- How does pH range affect the function and structure of catecholoxidase?
Help the enzymes functionbetter
- The stomach maintains a pH of approximately 3 to 5. Do allenzymes function at this pH level? Examine the data in Exercise 4and determine if catechol oxidase would function in the stomach.
No, most enzymes functionbetter at ph 6-8
- What is enzyme specificity? Give an example.
A specific substrate fits aspecific enzyme.