Enzymes

Introduction

Click here to go to the section on Energy and Enzymes in the Bio 101 lecture notes.

What are Enzymes?

Catalysts are substances that speed up chemical reactions.  Organic catalysts are called enzymes.

Reactions with enzymes are up to 10 billion times faster than those without enzymes. 

Enzymes are specific for one particular reaction or group of related reactions.

Many reactions cannot occur without the correct enzyme present.

An enzyme-substrate complex forms when the enzyme’s active site binds with the substrate like a key fitting a lock. 

The shape of the enzyme must match the shape of the substrate. Enzymes are therefore very specific; they will only function correctly if the shape of the substrate matches the active site.

The enzyme does not form a chemical bond with the substrate. After the reaction, the products are released and the enzyme returns to its normal shape.

Because the enzyme does not form chemical bonds with the substrate, it remains unchanged. As a result, the enzyme molecule can be reused. Only a small amount of enzyme is needed because they can be used repeatedly.

Laboratory Exercise

This laboratory exercise will explore the effect of temperature, pH, and enzyme concentration on the rate of a reaction.

Part A- Temperature

Higher temperature generally causes more collisions among the molecules and therefore increases the rate of a reaction. More collisions increase the likelihood that substrate will collide with the active site of the enzyme, thus increasing the rate of an enzyme-catalyzed reaction.

Above a certain temperature, activity begins to decline because the enzyme begins to denature.

The rate of chemical reactions therefore increases with temperature but then decreases as enzymes denature.

Rennin is an enzyme found in the stomach of mammals where it functions to solidify milk. You will observe this enzyme functioning by mixing it with milk in a test tube.

In this section, you will explore the effect of cold temperatures and warm temperatures on the rate of reaction. The presence of a reaction is indicated by milk becoming solid.

A1. Predict what will happen in each of the tubes described in the experiment below and write your hypotheses on the answer sheet. You should have two hypotheses- one that addresses the the effect of temperature on rate of reaction (tubes 1 and 3) and one that addresses the effect of denaturing the enzyme on reaction time (tubes 3 and 5). Your hypotheses should be specific for this experiment; it should state the expected outcome of this experiment.

Be sure to write your hypotheses as statements, not as questions. At this point, you do not know what will happen; your hypotheses may be correct or incorrect. An incorrect hypothesis is perfectly acceptable. You will test the hypotheses by performing the experiment below.

A2. Obtain five test tubes and put a mark 2 cm from the bottom of each using a wax pencil. 

A3. Mark the tubes with your initials or some other means to identify them. The tubes should also be numbered 1 through 5.

A4. Fill all of the tubes to the 2 cm mark with milk.

A5. Add 3 drops of rennin to the milk in tube #1 and keep it at room temperature for 15 minutes. Tube #2 without rennin should also be kept at room temperature to serve as a control.

A6. Add 3 drops of rennin to the milk in tube #3 and place it in a 37 degree incubator for 15 minutes. Tube #4 without rennin should also be placed in the incubator for 15 minutes to serve as a control.

A7. Add 3 drops of boiled rennin to the milk in tube #5 and place it in the 37 degree incubator for 15 minutes along with the tubes prepared in step #6 above. If boiled rennin is not available, put a small amount of rennin in a beaker and bring the rennin to a boil by placing the beaker on a hotplate. The rennin will be ready to use after it has begun to boil.

A8. Observe the milk in each of the five tubes after the 15 minute period and record your observations in table 1 on the answer sheet. 

A9. Do your results support either of your hypotheses? Explain.

Results

Top tube - Cold milk and rennin, 15 minutes Bottom tube - Control Both tubes were incubated in ice water for 15 minutes. Click on the image to view an enlargement.
Top tube - Warm milk and rennin Bottom tube - Control Both tubes were incubated at 37 degrees C for 15 minutes.  Click on the image to view an enlargement.
Top tube - Warm milk and previously-boiled rennin Bottom tube - Control Both tubes were incubated at 37 degrees C for 15 minutes.  Click on the image to view an enlargement.

Part B- pH

Each enzyme has an optimal pH.

A change in pH can alter the ionization of the R groups of the amino acids. When the charges on the amino acids change, hydrogen bonding within the protein molecule change and the molecule changes shape. The new shape may not be effective.

The diagram below shows that pepsin functions best in an acid environment. This makes sense because pepsin is an enzyme that is normally found in the stomach where the pH is low due to the presence of hydrochloric acid. Trypsin is found in the duodenum, and therefore, its optimum pH is in the neutral range to match the pH of the duodenum.

Most cells form hydrogen peroxide (H₂O₂) as a waste product of aerobic respiration. Hydrogen peroxide is toxic and must be converted to water and oxygen by the enzyme catalase.

Hydrogen peroxide is also commonly used as a household disinfectant. It bubbles when it is applied to cuts and scrapes because catalase is present in the fluids of the broken cells. As the equation above shows, the bubbles are oxygen gas (O₂).

In the experiment below, bubbling will be used as an indication that a reaction is occurring. A faster reaction will have more bubbling. 

B1. Predict how pH will affect the rate of reaction in the three different situations described below and write your hypothesis or hypotheses on the answer sheet. At this point, you do not know if your hypothesis is true or false. It is acceptable to create a hypothesis which will be shown to be false.

B2. Mark three test tubes with a wax pencil 3 cm from the bottom and 6 cm from the bottom.

B3. Add 3 cm of hydrogen peroxide to each tube.

B4. Fill one of the tubes to the 6 cm mark with 6 m HCl.

B5. Fill another tube to the 6 cm mark with 5 m NaOH.

B6. Fill the third tube to the 6 cm mark with distilled water.

B7. Cut three pieces of potato that are one cubic centimeter (cc) each.

B8.  Add one of the potato cubes to each of the 3 tubes that you prepared in steps 2 - 6 above. It may be helpful to use a small spatula when transferring the potato to the tube.

B9. Write your observation of the amount of bubbling in table 2. We are only interested in the amount of bubbling. We are not interested in any change in color or whether the potato floats or sinks.

B10. Use pH paper to measure the pH of each tube and record your measurements in table 2 on the answer sheet.

B11. At what pH does catalase function best?

B12. Do your results support your hypothesis? Explain.

B13. The pH of the stomach is normally about 2.0. At what pH do you predict rennin works best? (Hint: Rennin is found in the stomach of mammals).

B14. What can you say about pH and enzyme functioning? Is there a single pH that enzymes function best at or does it depend on the enzyme?

Part C- Substrate Concentration

At lower concentrations, the active sites on most of the enzyme molecules are not filled because there is not much substrate.  Higher concentrations cause more collisions between the molecules.  With more molecules and collisions, enzymes are more likely to encounter molecules of reactant.

The maximum velocity of a reaction is reached when the active sites are almost continuously filled. Increased substrate concentration after this point will not increase the rate.  Reaction rate therefore increases as substrate concentration is increased but it levels off.

Enzyme Concentration

If there is insufficient enzyme present, the reaction will not proceed as fast as it otherwise would because there is not enough enzyme for all of the reactant molecules. As the amount of enzyme is increased, the rate of reaction increases. If there are more enzyme molecules than are needed, adding additional enzyme will not increase the rate. Reaction rate therefore increases as enzyme concentration increases but then it levels off.

In this investigation, we will examine what happens to the rate of a reaction when the amount of enzyme is reduced. We will use urease, an enzyme that converts urea to ammonia. The ammonia causes the pH of the water to increase (it becomes more basic). You will be able to tell when a reaction occurs because the urea solution also contains a pH indicator that is becomes yellow in acid but turns red when the solution becomes basic.

The object of this experiment is to measure the amount of time it takes for the solution to turn red if less enzyme is used.

C1. Create a hypothesis regarding the the amount of urease and the rate of reaction of Urea.

C2. Obtain four test tubes and add 2 cm of urea to each.

C3. Label three of these tubes 1 through 3; the remaining tube will not be used; it will serve as a control.

C4. Have your lab partner start timing as you add 15 drops of urease to tube #1 and then swirl the tube until it changes to a red color. Record the amount of time that it took for the urease to change to a red color.

C5. Add 5 drops of urease to tube #2 and then swirl the tube until it changes to a red color. Record the amount of time that it took for the urease to change to a red color.

C6. Add 1 drop of urease to tube #3 and then swirl the tube until it changes to a red color. Record the amount of time that it took for the urease to change to a red color.

C7. Record your results in the answer sheet.

C8. Did using less enzyme produce a reaction?

C9. What was the effect of using less enzyme in your experiment? If your experiment did not work as expected, what should have happened?

C10. In general, what happens to the rate of reaction as the amount of enzyme is decreased?

C11. Do your results support your hypothesis? Explain.