ENZYME CATALYSIS
Background
For a chemical reaction to occur, reactants must collide with sufficient force to break their chem- ical bonds, allowing new bonds to be formed between the products. All chemical reactions in living organisms would be too slow to sustain life without specialized substances called enzymes. Enzymes act as catalysts to speed up chemical reactions by lowering the activation energy of
a reaction (Figure 1). Activation energy is the minimum amount of energy required to start a chemical reaction.
Figure 1.
Activation energy is present when kinetic energy drives reactants to a level where their random collisions are strong enough to ensure the interaction in such a way that the reactants’ bonds break and are rearranged into prod- ucts. Enzymes work by stabilizing the transition state of the substrate, allowing it to moveto
the product state. In other words, the enzyme creates an environment that allows the reac- tion to proceed. Enzymes and their cognate substratesfita“lock-and-key”model.Onevery enzyme,thereisabindingsiteshapedtofita
specific substrate. This allows enzymes to work with high efficiency and specificity. Like all cata- lysts, the enzyme is not consumed in the reac- tion and can be used repeatedly.
Figure 2.
Enzymes in living organisms operate best within a narrow range of environmental condi- tions (e.g., temperature, concentration, pH, etc.). For example, the human digestive system utilizes several enzymes to break downfood.
Different enzymes operate in different partsof
the digestive tract. Some parts of the digestive tract are acidic (pH less than 7), whereas others are alkaline or basic (pH greater than 7). The stomach contains acidic gastric juices that aid digestion. Enzymes active in the stomach func- tion best in that acidic environment. When food exits the stomach it enters the small intestine, which provides a more alkaline environment so the acidic food coming from the stomach can be neutralized. Enzymes active in the small intes- tine function best in a weakly alkaline environ- ment. When pH values are outside the optimum range for digestive enzymes, denaturing of the molecule or alteration of the active site shape can occur so that the enzyme cannot accept the substrate. Enzymes active in the stomachare
continued on next page
ENZYME CATALYSIS
Materials
Included in the materials kit:
Yeast (source of catalase)
2 Plastic dropping pipets
- Medicine
cups, 1 oz
- Filterpapers
Petri dish, 60 × 15 mm
Needed from theequipmentkit: Needed, but notsupplied:
- Hydrogen peroxide,3%*
- Water, bottled orpurified
- Access to tap water (hot andcold)
- Single-hole punch
- Teaspoon
- Timer (stopwatch, cell phone, online timer),accurate to 0.1seconds
- Crushed iceor small icecubes
Forceps
Ruler
Graduated cylinder, 25 mL
5 Test tubes,
17 × 100 mm
Test tube rack
Grease pencil
- Plastic trash bag or oldtowel
- Calculator
- Graph paper or graphingsoftware
Thermometer Beaker, 250mL
ENZYME CATALYSIS
Safety
Wear your safety goggles, gloves, and
lab apron at all times while conducting this investigation.
Read all the instructions for this laboratory activity before beginning. Follow the instructions closely, and observe established laboratory safety practices, including the use of appro- priate personal protective equipment (PPE) as described in the Safety and Procedure sections.
Use caution with 3% hydrogen peroxide: It is a strong oxidizer. Use near a source of running water that can be used as a safety eye wash or safety shower if any hydrogen peroxide comes in contact withskin
or eyes. Hydrogen peroxide will bleach colored fabrics. If spilled on clothing, immediately rinse with water. All wastes from this activity are safe to wash down the drain.
Do not eat, drink, or chew gum while performing this activity. Wash your hands with soap and water before and after performing the activity.
Clean up the work area with soap and water after completing the investigation. Keep pets and children away from lab materials and equipment.
Preparation
- Read through the Procedure.
- Using the single-hole punch, punch out 20 disks from a piece of filter paper into the open Petri dish. If you do not have a hole punch, cut the filter paper into small squares (6–7 mm). These are called filter paper disks
- Obtain materials and place them on a counter covered by a plastic trash bag or anold
towel to prevent the hydrogen peroxide from discoloring the counter surface.
- Turn on a hot water tap and monitor the temperature with the thermometer until a temperature of 35 °C is achieved (or use a microwave to heat water in a glass cup. Do not use the plastic beaker for microwave heating!). Collect approximately 250 mL of the 35 °C water in the
- Measure a level teaspoon of dry yeast, add it to the 35 °C water, and stir thoroughly with the teaspoon. Consider this the100%
catalase solution to be used for the remainder of the lab.
- Using the grease pencil, write “100% catalase”ononeofthemedicine
- Pour the yeast suspension into the medicine cup up to the 20.0-mLmark.
- Label the bulb of one pipet “water” (or W) and the other pipet “catalase” (or C) with the greasepencil.
ACTIVITY
ACTIVITY 1
Enzyme Concentration
- Using the grease pencil, label the empty medicine cup “1.5%H2O2.”
- Measure 10 mL of 3% hydrogen peroxide using the 25-mL graduated cylinder and add it to the “1.5% H2O2” medicine cup. Also add 10 mL of room-temperature bottledwater
to the medicine cup to yield 20 mL of 1.5% hydrogen peroxide. This will be the reaction vessel.
- Using the grease pencil (or a permanent maker), label five test tubes with 0%, 25%, 50%, 75%, and 100%. These labels represent the concentrations of catalase that will be used in eachtube.
- (1 ml equals to about 20 drops). Take the pipet labeled “catalase” and add the following drops of catalase to each test tube except for the control, as shown below in the table. Take the pipet labeled “water” and add enough drops of bottled water to obtain a final volume of 20 drops (1 mL) per test tube as shown below in the table.
| 0% (control) | 25% | 50% | 75% | 100% |
| 0 drops catalase | 5 drops catalase | 10 drops catalase | 15 drops catalase | 20 drops catalase |
| 20 drops water | 15 drops water | 10 drops water | 5 drops water | 0 drops water |
- Swirl each of the five test tubes to mix the
- With a pair of forceps, take a dry filter disk out of the Petri dish and dip it into the 0% test tube solution. Tilt the test tube to allow the liquid to run down to the disk. Remove the disk and blot by dragging it across the top of the test tube on bothsides.
- Start the timer as soon as the disk is placedwithforcepsonthebottomof
the medicine cup containing 20 mL 1.5% hydrogen peroxide. Time in seconds how long it takes the disk to rise to the surface of the peroxide. This is the control data point. Stop timing after 3 minutes if there is no activity. Record the time (in seconds) in Data Table 1.
- Remove the used disk. Perform an additional trial with a new soaked disk from the control, 0% catalase.
- Conduct two trials for each of the remaining catalase solutions (25%, 50%, 75%, and 100%). Swirl each test tube to mix the solution before wetting the disk. If the two trials vary by more than 4 seconds, run a third trial and average the two closest times in seconds. Start with the lowest percent concentration of catalase (25%) and move to each higher concentration. Record the time in seconds in Data Table
- For each concentration, average the two times for floating the disks. Always remove the floating disk with forceps before starting the next trial.
continued on next page
ACTIVITY
ACTIVITY1continued ACTIVITY2
- Measure the height of the 1.5% hydrogen peroxide solution to the nearest 0.1 cm. This is the distance traveled by the disk from the bottom to the surface of the peroxide, and it will be the same for all trials where the disk
- The rate of each reaction can be calculated by the expression R = d/t, where d = the distance in cm that the disk travels from the bottom of the container to the surface, and t = average time in seconds. The unit for the rate is cm/s. If the disk remains on the bottom of the container after 3 minutes, the distance traveled (d) will be zero, and the rate will be zero.
- Plot a graph for each data point with rate on the y-axis and percent concentrationof
enzyme on the x-axis. If the points are linear, draw a “best-fit” straight line through or near all of the data points. Alternatively, use a computerized spreadsheet with graphing and select “best fit”line.
- If Activity 2 will be performed immediately, keep the 1.5% hydrogen peroxide and 100% catalase in their respective medicine cups and ignore preparation steps 1 and 2 in Activity 2. If Activity 2 will be performedat
a later time, empty both medicine cups in a sink and rinse with water. All five test tubes should be thoroughly washed and dried. It is safe to empty and rinse the test tubes down
the sink drain.
Effect of Temperature
- If more than an hour has passed since completing Activity 1, prepare a fresh solution of 1.5% hydrogen peroxide by adding 10 mL water to 10 mL 3% hydrogen peroxide in the same medicine cup labeled 1.5%H2O2.
- If more than an hour has passed since completing Activity 1, prepare another batch of 100% yeast solution (one teaspoon per cup of warm, 35 °C water, and mix Pour into the medicine cup labeled 100% catalase up to the 20.0-mLmark.
- Using the grease pencil, re-label the five test tubes, place them in the test tube rack, and add 20 drops of the 100% catalase with the designated pipet into each testtube.
- Measure the room temperature in °C and record the value in the heading of Data Table 2 and on one of the 5 testtubes.
- Record the temperature that will be 10 °C below room temperature on the second tube. On the third, record 10 °C above room temperature, and on the others 20 °Cand
30 °C above room temperature. For example, if temperature is 25 °C, then the tubes would be labeled 15 °C, 25 °C, 35 °C, 45 °C, and 55 °C. Fill in the actual temperatures in the heading columns of Data Table2.
- Soak a filter disk in room temperature catalase, blot, and place it usingthe
forceps at the bottom of the 1.5% H O cup.
2 2
Time how long it takes for the disk to rise to the surface, and record the value in Data Table 2.
continued on next page
- For the below room temperature, set up an ice bath by adding an inch of crushed ice to the
- Place the medicine cup containing 1.5% hydrogen peroxide in the center of the ice and insert the catalase test tube labeled 10 °C below in the ice beside thecup.
- Monitor the temperature of the medicine cup by placing the thermometer in the peroxide solution. Remove the cup when the temperature decreases to 10 °C belowroom
temperature. The assumption is made that the temperature of the catalase in the test tube is at the same temperature.
- Soak a filter disk in the cooled catalase, blot it on the test tubemouth,
and place it on the bottom of the medicine cup. Record how long it takes the disk to rise to the surface.
- Discard the ice.
- For temperatures above room temperature, set up a water bath by adding hot tap water to the Insert a thermometer into the water bath to track the temperature. Adjust the temperature by replacing some of the hot water with cooler tap water until the desired temperature is achieved. The total volume of water in the beaker should be approximately 50mL.
- Place the medicine cup of hydrogen peroxide in the center of the Place the catalase tube labeled 10 °C above room temperature in the beaker beside the medicine cup. Let it stand 2 minutesto
allow the temperature in the medicine cup to equilibrate with the water bath.
- Soakafilterdiskinthewarmcatalase, blotit,andplaceonthebottomofthe
medicine cup. Record how long it takes the disk to rise to the surface.
- Repeat steps 12–14 for 20 and 30 °C above room temperature and record the times and temperatures in Data Table
- Calculate the rate (d/t) for each temperature and record the values in Data Table
- Plot a graph for each data point with rate on the y-axis and temperature on thex-axis.
ACTIVITY
Data Table 1: Catalase Enzyme Concentration
| 0% | 25% | 50% | 75% | 100% | |
| Depth of H2O2 Solution | |||||
| Trial 1 Time | |||||
| Trial 2 Time | |||||
| Avg. Time | |||||
| Rate (d/t) |
Data Table 2: Effect of Temperature on Catalase Enzyme
| 10°
Below |
Rm. Temp. | 10°
Above |
20°
Above |
30°
Above |
|
| Depth of H2O2 Solution | |||||
| Time | |||||
| Rate (d/t) |
Disposal and Cleanup
- Dispose of solutions down the drain with the water running. Allow the faucet to run a few minutes to dilute thesolutions.
- Wipe up any hydrogen peroxide spills, as they could bleach colored fabrics, counter tops, and clothing.
- Rinse and dry the lab equipment and return the materials to your equipmentkit.
- Sanitize the workspace.
Summary
Summarize your major results and upload images.
Background
For a chemical reaction to occur, reactants must collide with sufficient force to break their chem- ical bonds, allowing new bonds to be formed between the products. All chemical reactions in living organisms would be too slow to sustain life without specialized substances called enzymes. Enzymes act as catalysts to speed up chemical reactions by lowering the activation energy of
a reaction (Figure 1). Activation energy is the minimum amount of energy required to start a chemical reaction.
Figure 1.
Activation energy is present when kinetic energy drives reactants to a level where their random collisions are strong enough to ensure the interaction in such a way that the reactants’ bonds break and are rearranged into prod- ucts. Enzymes work by stabilizing the transition state of the substrate, allowing it to moveto
the product state. In other words, the enzyme creates an environment that allows the reac- tion to proceed. Enzymes and their cognate substratesfita“lock-and-key”model.Onevery enzyme,thereisabindingsiteshapedtofita
specific substrate. This allows enzymes to work with high efficiency and specificity. Like all cata- lysts, the enzyme is not consumed in the reac- tion and can be used repeatedly.
Figure 2.
Enzymes in living organisms operate best within a narrow range of environmental condi- tions (e.g., temperature, concentration, pH, etc.). For example, the human digestive system utilizes several enzymes to break downfood.
Different enzymes operate in different partsof
the digestive tract. Some parts of the digestive tract are acidic (pH less than 7), whereas others are alkaline or basic (pH greater than 7). The stomach contains acidic gastric juices that aid digestion. Enzymes active in the stomach func- tion best in that acidic environment. When food exits the stomach it enters the small intestine, which provides a more alkaline environment so the acidic food coming from the stomach can be neutralized. Enzymes active in the small intes- tine function best in a weakly alkaline environ- ment. When pH values are outside the optimum range for digestive enzymes, denaturing of the molecule or alteration of the active site shape can occur so that the enzyme cannot accept the substrate. Enzymes active in the stomachare
continued on next page
ENZYME CATALYSIS
Materials
Included in the materials kit:
Yeast (source of catalase)
2 Plastic dropping pipets
- Medicine
cups, 1 oz
- Filterpapers
Petri dish, 60 × 15 mm
Needed from theequipmentkit: Needed, but notsupplied:
- Hydrogen peroxide,3%*
- Water, bottled orpurified
- Access to tap water (hot andcold)
- Single-hole punch
- Teaspoon
- Timer (stopwatch, cell phone, online timer),accurate to 0.1seconds
- Crushed iceor small icecubes
Forceps
Ruler
Graduated cylinder, 25 mL
5 Test tubes,
17 × 100 mm
Test tube rack
Grease pencil
- Plastic trash bag or oldtowel
- Calculator
- Graph paper or graphingsoftware
Thermometer Beaker, 250mL
ENZYME CATALYSIS
Safety
Wear your safety goggles, gloves, and
lab apron at all times while conducting this investigation.
Read all the instructions for this laboratory activity before beginning. Follow the instructions closely, and observe established laboratory safety practices, including the use of appro- priate personal protective equipment (PPE) as described in the Safety and Procedure sections.
Use caution with 3% hydrogen peroxide: It is a strong oxidizer. Use near a source of running water that can be used as a safety eye wash or safety shower if any hydrogen peroxide comes in contact withskin
or eyes. Hydrogen peroxide will bleach colored fabrics. If spilled on clothing, immediately rinse with water. All wastes from this activity are safe to wash down the drain.
Do not eat, drink, or chew gum while performing this activity. Wash your hands with soap and water before and after performing the activity.
Clean up the work area with soap and water after completing the investigation. Keep pets and children away from lab materials and equipment.
Preparation
- Read through the Procedure.
- Using the single-hole punch, punch out 20 disks from a piece of filter paper into the open Petri dish. If you do not have a hole punch, cut the filter paper into small squares (6–7 mm). These are called filter paper disks
- Obtain materials and place them on a counter covered by a plastic trash bag or anold
towel to prevent the hydrogen peroxide from discoloring the counter surface.
- Turn on a hot water tap and monitor the temperature with the thermometer until a temperature of 35 °C is achieved (or use a microwave to heat water in a glass cup. Do not use the plastic beaker for microwave heating!). Collect approximately 250 mL of the 35 °C water in the
- Measure a level teaspoon of dry yeast, add it to the 35 °C water, and stir thoroughly with the teaspoon. Consider this the100%
catalase solution to be used for the remainder of the lab.
- Using the grease pencil, write “100% catalase”ononeofthemedicine
- Pour the yeast suspension into the medicine cup up to the 20.0-mLmark.
- Label the bulb of one pipet “water” (or W) and the other pipet “catalase” (or C) with the greasepencil.
ACTIVITY
ACTIVITY 1
Enzyme Concentration
- Using the grease pencil, label the empty medicine cup “1.5%H2O2.”
- Measure 10 mL of 3% hydrogen peroxide using the 25-mL graduated cylinder and add it to the “1.5% H2O2” medicine cup. Also add 10 mL of room-temperature bottledwater
to the medicine cup to yield 20 mL of 1.5% hydrogen peroxide. This will be the reaction vessel.
- Using the grease pencil (or a permanent maker), label five test tubes with 0%, 25%, 50%, 75%, and 100%. These labels represent the concentrations of catalase that will be used in eachtube.
- (1 ml equals to about 20 drops). Take the pipet labeled “catalase” and add the following drops of catalase to each test tube except for the control, as shown below in the table. Take the pipet labeled “water” and add enough drops of bottled water to obtain a final volume of 20 drops (1 mL) per test tube as shown below in the table.
| 0% (control) | 25% | 50% | 75% | 100% |
| 0 drops catalase | 5 drops catalase | 10 drops catalase | 15 drops catalase | 20 drops catalase |
| 20 drops water | 15 drops water | 10 drops water | 5 drops water | 0 drops water |
- Swirl each of the five test tubes to mix the
- With a pair of forceps, take a dry filter disk out of the Petri dish and dip it into the 0% test tube solution. Tilt the test tube to allow the liquid to run down to the disk. Remove the disk and blot by dragging it across the top of the test tube on bothsides.
- Start the timer as soon as the disk is placedwithforcepsonthebottomof
the medicine cup containing 20 mL 1.5% hydrogen peroxide. Time in seconds how long it takes the disk to rise to the surface of the peroxide. This is the control data point. Stop timing after 3 minutes if there is no activity. Record the time (in seconds) in Data Table 1.
- Remove the used disk. Perform an additional trial with a new soaked disk from the control, 0% catalase.
- Conduct two trials for each of the remaining catalase solutions (25%, 50%, 75%, and 100%). Swirl each test tube to mix the solution before wetting the disk. If the two trials vary by more than 4 seconds, run a third trial and average the two closest times in seconds. Start with the lowest percent concentration of catalase (25%) and move to each higher concentration. Record the time in seconds in Data Table
- For each concentration, average the two times for floating the disks. Always remove the floating disk with forceps before starting the next trial.
continued on next page
ACTIVITY
ACTIVITY1continued ACTIVITY2
- Measure the height of the 1.5% hydrogen peroxide solution to the nearest 0.1 cm. This is the distance traveled by the disk from the bottom to the surface of the peroxide, and it will be the same for all trials where the disk
- The rate of each reaction can be calculated by the expression R = d/t, where d = the distance in cm that the disk travels from the bottom of the container to the surface, and t = average time in seconds. The unit for the rate is cm/s. If the disk remains on the bottom of the container after 3 minutes, the distance traveled (d) will be zero, and the rate will be zero.
- Plot a graph for each data point with rate on the y-axis and percent concentrationof
enzyme on the x-axis. If the points are linear, draw a “best-fit” straight line through or near all of the data points. Alternatively, use a computerized spreadsheet with graphing and select “best fit”line.
- If Activity 2 will be performed immediately, keep the 1.5% hydrogen peroxide and 100% catalase in their respective medicine cups and ignore preparation steps 1 and 2 in Activity 2. If Activity 2 will be performedat
a later time, empty both medicine cups in a sink and rinse with water. All five test tubes should be thoroughly washed and dried. It is safe to empty and rinse the test tubes down
the sink drain.
Effect of Temperature
- If more than an hour has passed since completing Activity 1, prepare a fresh solution of 1.5% hydrogen peroxide by adding 10 mL water to 10 mL 3% hydrogen peroxide in the same medicine cup labeled 1.5%H2O2.
- If more than an hour has passed since completing Activity 1, prepare another batch of 100% yeast solution (one teaspoon per cup of warm, 35 °C water, and mix Pour into the medicine cup labeled 100% catalase up to the 20.0-mLmark.
- Using the grease pencil, re-label the five test tubes, place them in the test tube rack, and add 20 drops of the 100% catalase with the designated pipet into each testtube.
- Measure the room temperature in °C and record the value in the heading of Data Table 2 and on one of the 5 testtubes.
- Record the temperature that will be 10 °C below room temperature on the second tube. On the third, record 10 °C above room temperature, and on the others 20 °Cand
30 °C above room temperature. For example, if temperature is 25 °C, then the tubes would be labeled 15 °C, 25 °C, 35 °C, 45 °C, and 55 °C. Fill in the actual temperatures in the heading columns of Data Table2.
- Soak a filter disk in room temperature catalase, blot, and place it usingthe
forceps at the bottom of the 1.5% H O cup.
2 2
Time how long it takes for the disk to rise to the surface, and record the value in Data Table 2.
continued on next page
- For the below room temperature, set up an ice bath by adding an inch of crushed ice to the
- Place the medicine cup containing 1.5% hydrogen peroxide in the center of the ice and insert the catalase test tube labeled 10 °C below in the ice beside thecup.
- Monitor the temperature of the medicine cup by placing the thermometer in the peroxide solution. Remove the cup when the temperature decreases to 10 °C belowroom
temperature. The assumption is made that the temperature of the catalase in the test tube is at the same temperature.
- Soak a filter disk in the cooled catalase, blot it on the test tubemouth,
and place it on the bottom of the medicine cup. Record how long it takes the disk to rise to the surface.
- Discard the ice.
- For temperatures above room temperature, set up a water bath by adding hot tap water to the Insert a thermometer into the water bath to track the temperature. Adjust the temperature by replacing some of the hot water with cooler tap water until the desired temperature is achieved. The total volume of water in the beaker should be approximately 50mL.
- Place the medicine cup of hydrogen peroxide in the center of the Place the catalase tube labeled 10 °C above room temperature in the beaker beside the medicine cup. Let it stand 2 minutesto
allow the temperature in the medicine cup to equilibrate with the water bath.
- Soakafilterdiskinthewarmcatalase, blotit,andplaceonthebottomofthe
medicine cup. Record how long it takes the disk to rise to the surface.
- Repeat steps 12–14 for 20 and 30 °C above room temperature and record the times and temperatures in Data Table
- Calculate the rate (d/t) for each temperature and record the values in Data Table
- Plot a graph for each data point with rate on the y-axis and temperature on thex-axis.
ACTIVITY
Data Table 1: Catalase Enzyme Concentration
| 0% | 25% | 50% | 75% | 100% | |
| Depth of H2O2 Solution | |||||
| Trial 1 Time | |||||
| Trial 2 Time | |||||
| Avg. Time | |||||
| Rate (d/t) |
Data Table 2: Effect of Temperature on Catalase Enzyme
| 10°
Below |
Rm. Temp. | 10°
Above |
20°
Above |
30°
Above |
|
| Depth of H2O2 Solution | |||||
| Time | |||||
| Rate (d/t) |
Disposal and Cleanup
- Dispose of solutions down the drain with the water running. Allow the faucet to run a few minutes to dilute thesolutions.
- Wipe up any hydrogen peroxide spills, as they could bleach colored fabrics, counter tops, and clothing.
- Rinse and dry the lab equipment and return the materials to your equipmentkit.
- Sanitize the workspace.
Summary
Summarize your major results and upload images.
