Enzymes are organic catalysts. They increase the speed of a chemical reaction without themselves undergoing any permanent chemical change. They are neither used up in the reaction nor do they appear as reaction products. The basic enzymatic reaction can be represented as a two step process:
S
+ E ES
ES P+ E
where E represents the enzyme catalyzing the reaction, S the substrate (the substance being changed), and P the product(s) of the reaction. ES is an intermediate enzyme-substrate complex formed by the reaction of enzyme with substrate. This complex quickly disassociates upon alteration of the substrate to release the enzyme and the reaction product(s).
All organisms are capable of carrying out an amazing range of complex chemical reactions. Organic compounds can be built up, broken down, or otherwise altered. Only by the use of enzymes can these reactions occur at the rate, degree of efficiency, temperature, and pH found in living cells.
All presently known enzymes are proteins, consisting of one or more chains of polypeptides. Each enzyme molecule has a particular three-dimensional shape. This shape can be altered or sometimes irreversibly disrupted by heat, pH changes, or the presence of certain salts or metal ions. At a given pH, an enzyme molecule has areas of + and - charge as a result of ionization of some of its amino acids. As pH changes, the distribution of electrical charge within the enzyme molecule also changes.
An enzyme’s activity is determined by the three-dimensional structure of its molecule and the distribution of electrical charge on the molecule. Somewhere on the enzyme molecule is a pocket (the active site) into which the substrate molecule fits. Within this active site there must be a correspondence of electrical charge between the enzyme molecule and the substrate. Thus, the substrate molecule must be of a correct shape and have a correct distribution of electrical charge if it is to be acted upon by a given enzyme. This means that each enzyme is specific for a particular substrate molecule or class of substrate molecules.
In some cases there are molecules that are similar in size and shape to the true substrate molecule. These impostors (called inhibitors) may fit into the enzyme’s active site even though they are not acted upon. Although the inhibitor molecule occupies the active site for only a brief period, while there it prevents the substrate molecule from entering, thus slowing the rate of enzyme action. This is termed competitive inhibition.
Noncompetitive inhibition can result when substances combine with the enzyme to form compounds in the active site or change the distribution of charge in the active site. Many substances can combine with enzymes in this way. Heavy metals such as silver, mercury, and lead are inhibitors of many enzymes.
Preparation of Solutions and Lab Materials
Deionized or distilled water must be used in the preparation of all solutions. Wash all glassware and rinse three times with deionized or distilled water before using. This will cleanse the glassware of any alkali traces. Dirty glassware can cause the bromothymol blue to change color prematurely.
Before beginning any exercise, obtain three pipets. Label one “enzyme” to be used with the enzyme solutions only. Label one pipet “substrate” and use it for substrate solutions only. The other pipet is used for all solutions other than enzymes or substrates. When a pipet has been used for one solution, rinse it three times inside and outside before using it with another solution. Always wash all glassware at the end of a lab session.
All solutions should be refrigerated between lab sessions. Allow the solutions to come to room temperature before using. Make up the solutions only as needed. The directions which follow are for classroom quantities of the solutions.
Buffer Solutions
Buffer pH 7. Dissolve the powder contained in a pH 7 capsule in 100 ml of water.
Buffer pH 4. Dissolve the powder contained in a pH 4 capsule in 100 ml of water.
Buffer pH 9. Dissolve the powder contained in a pH 9 capsule in 100 ml of water.
Buffer pH 5.9. Mix 4 ml of pH 7 buffer solution with 2 ml of pH 4 buffer solution.
Buffer pH 6.5. Mix 4 ml of pH 7 buffer solution with 1 ml of pH 4 buffer solution.
Buffer pH 7.9. Mix 3 ml of pH 7 buffer solution with 3 ml of pH 9 buffer solution.
Buffer pH 8.3. Mix 2 ml of pH 7 buffer solution with 3 ml of pH 9 buffer solution.
Urease Solution
Weigh out 1 g of urease (Jack bean meal). Add the urease to 100 ml of pH 7 buffer. Shake or stir the solution gently but thoroughly for one minute. Allow the solution to stand for five minutes, then shake or stir again for one minute. Filter the solution through a filter paper. When stored in a refrigerator, the solution will retain its activity for about two days. This solution can be remade once, if needed.
Mix 2 ml of urease solution with 6 ml of pH 7 buffer solution.
Mix 2 ml of 0.75 M urea solution with 18 ml of water.
Mix 2 ml of 0.75 M urea solution with 98 ml of water.
Open the thiourea packet and dissolve the contents in 30 ml of water. Rinse out the packet with some of the solution to be sure all the thiourea is dissolved.
Mix 3 ml of 0.75 M urea solution with 9 ml of 1.0 M thiourea solution.
Mix 3 ml of 0.75 M urea solution with 6 ml of water and 3 ml of 1.0 M thiourea solution.
Mix 3 ml of 0.75 M urea with 9 ml of water.
Use undiluted from the container.
Mix 1 ml of concentrated bacterial amylase with 40 ml of water.
EXERCISES
Part I: Urease Activity and Use of the Diffusion Dish
Solutions needed: Urease, 0.75 M urea, bromothymol blue.
Urease hydrolyses urea to form carbon dioxide and ammonia gas. We will use bromothymol blue to detect the presence of the ammonia which results from the hydrolysis of urea.
Obtain a diffusion dish and set it on a white surface. Label one side “A” and the other side “B.”
Pipet 2 ml of bromothymol blue into side A of dish. Pipet 2 ml of 0.75 M urea into the B side. Start the reaction by pipeting 1 ml of urease solution into the B side of the dish. Immediately place the lid on the dish. Agitate the dish with a rotary motion to distribute the urease. Be careful that none of the bromothymol blue spills into the B side. Continue agitating the dish.
Bromothymol blue is blue at a basic pH, green at neutral pH, and yellow at an acid pH. The bromothymol blue supplied in the kit is acid and therefore yellow. As ammonia is produced by the reaction, it diffuses to the central chamber and dissolves in the bromothymol blue. Ammonia produces a basic solution. Observe when the bromothymol blue becomes a clear blue without any trace of green. We will use this as the end point. Empty the diffusion dish and rinse it three times with deionized or distilled water before reusing.
Solutions needed: Urease, 0.75 M Urea, 1.0 M thiourea, bromothymol blue, and iodine-potassium iodide.
Three teams should work together to complete this exercise.
Team A. Pipet 2 ml of bromothymol blue into the side A of the dish. Pipet 2 ml of 0.75M urea solution into side B of the dish, then pipet 1 ml of urease solution into side B. Replace the lid on the dish, note the time, and begin agitating the dish with a rotary motion. Record the time required for the bromothymol blue to change color.
Team B. Pipet 2 ml of bromothymol blue into side A of the dish. Pipet 2 ml of 0.75 M urea solution into side B of the dish. Add one drop of iodine-potassium iodide to side B, then pipet 1 ml of urease solution into side B. Replace the lid on the dish, note the time, and begin agitating the dish with a rotary motion. Record the time required for the bromothymol blue to change color.
Team C. Pipet 2 ml of bromothymol blue into side A of the dish. Pipet 2 ml of 1.0 M thiourea solution into side B of the dish, then pipet 1 ml of urease solution into side B. Replace the lid on the dish, note the time, and begin agitating the dish with a rotary motion. Continue your observations until teams A and B finish. Did the bromothymol blue change color?
Explain the results obtained by the three teams.
Solutions needed: 75:25 thiourea:urea, 50:50 thiourea:urea, 0:l00 thiourea:urea, urease, and bromothymol blue.
Three teams should work together to complete this exercise.
Team A. Pipet 2 ml of bromothymol blue into side A of the diffusion dish. Pipet 2 ml of 75:25 thiourea:urea solution into side B of the dish, then pipet 1 ml of urease into side B. Replace the lid on the dish, note the time, and begin agitating the dish with a rotary motor. Record the time required for the bromothymol blue to change color.
Team B. Pipet 2 ml of bromothymol blue into side A (the diffusion dish.) Pipet 2 ml of 50:50 thiourea:urea solution into side B of the dish, then pipet 1 ml of urease solution into side B. Replace the lid on the dish, note the time, and begin agitating the dish with a rotary motion. Record the time required for the bromothymol blue to change color.
Team C. Pipet 2 ml of bromothymol blue into side A of the diffusion dish. Pipette 2 ml of 0:100 thiourea:urea solution into side B of the dish, then pipet 1 ml of urease solution into side B. Replace the lid on the dish, note the time, and begin agitating the dish with a rotary motion. Record the time required for the bromothymol blue to change color.
Explain the results obtained by the three teams.
Solutions needed: 0.75 M urea, 0.075 M urea, 0.015 M urea, urease, and bromothymol blue.
Three teams should work together to complete this exercise.
Team A. Pipet 2 ml of bromothymol blue into side A of the dish. Into side B of the dish pipet 2 ml of 0.75 M urea, then pipet 1 ml of urease into side B. Replace the lid on the dish, note the time, and begin agitating the dish with a rotary motion. Record the time required for the bromothymol blue to change color.
Team B. Pipet 2 ml of bromothymol blue into side A of the dish. Into side B of the dish pipet 2 ml of 0.075 M urea solution, then pipet 1 ml of urease into side B. Replace the lid on the dish, note the time, and begin agitating the dish with a rotary motion. Record the time required for the bromothymol blue to change color.
Team C. Pipet 2 ml of bromothymol blue into side A of the dish. Into side B of the dish pipet 2 ml of 0.015 M urea solution, then pipet 1 ml of urease into side B. Replace the lid on the dish, note the time, and begin agitating the dish with a rotary motion. Record the time required for the bromothymol blue to change color.
Explain the results obtained by the three teams.
Solutions needed: Urease, half-strength urease, quarter-strength urease, 0.75 M urea, and bromothymol blue.
Three teams should work together to complete this exercise.
Team A. Pipet 2 ml of bromothymol blue into side A of the dish. Into side B of the dish pipet 2 ml of 0.75 M urea solution, then pipet 1 ml of urease into side B. Replace the lid on the dish, note the time, and begin agitating the dish with a rotary motion. Record the time required for the bromothymol blue to change color.
Team B. Pipet 2 ml of bromothymol blue into side A of the dish. Into side B of the dish pipet 2 ml of 0.75 M urea solution, then pipet 1 ml of half-strength urease solution into side B. Replace the lid on the dish, note the time, and begin agitating the dish with a rotary motion. Record the time required for the bromothymol blue to change color.
Team C. Pipet 2 ml of bromothymol blue into side A of the dish. Into side B of the dish pipet 2 ml of 0.75 M urea solution, then pipet 1 ml of quarter-strength urease solution into side B. Replace the lid on the dish, note the time, and begin agitating the dish with a rotary motion. Record the time required for the bromothymol blue to change color.
Explain the results obtained by the three teams.
Part II: Amylase Activity
Solutions needed: Amylose, dilute amylase, iodine-potassium iodide, and pH 7 buffer.
Obtain a reaction vial with dropper top. Mix in the vial 2 ml of amylose solution with 2 ml of pH 7 buffer. Add 1 ml of dilute amylase, note the time, cap the vial, and shake it to mix the contents. One minute after adding the dilute amylase, withdraw a sample and place three drops, one on top of the other, on a microscope slide or glass plate. Add one drop of iodine-potassium iodide to the sample on the slide or plate. Hold the slide or plate several centimeters above a white surface and observe the color of the sample. Dark blue-black indicates the presence of starch. Continue testing samples at one minute intervals until a dark brown color results. We will use this as the end point of the reaction. Record the time required for the reaction.
Solutions needed: pH 4 buffer, pH 5.9 buffer, pH 6.5 buffer, pH 7 buffer, pH 7.9 buffer, pH 8.3 buffer, pH 9 buffer, amylose, dilute amylase, and iodine-potassium iodide.
Three teams should work together to complete this exercise.
Team A: 2 ml pH 4 buffer, 2 ml amylose; 2 ml pH 5.9 buffer, 2 ml amylose; 2 ml pH 6.5 buffer, 2 ml amylose.
Team B: 2 ml pH 7 buffer, 2 ml amylose; 2 ml pH 7.9 buffer, 2 ml amylose; .
Team C: 2 ml pH 8.3 buffer, 2 ml amylose; 2 ml pH 9 buffer, 2 ml amylose.
Add 1 ml of dilute amylase to your vial, note the time, cap the vial, and shake it. Test samples at one minute intervals for the presence of starch. Record the time required for each team's reaction.
Using the data obtained, construct a graph. From your graph, determine the optimal pH for the amylase to act.
Solutions needed: Amylose, concentrated amylase, and iodine-potassium iodide.
Three teams should work together to complete this exercise.
Team A will use unheated concentrated amylase. Teams B and C will use concentrated amylase that has been heated in a boiling water bath as follows: Place 3 to 4 ml of concentrated amylase in a test tube and place the test tube in the boiling water bath.
Team B. Remove the test tube 1 after two minutes and cool in a cold water bath; Remove the test tube 2 after four minutes and cool in a cold water bath.
Team C. Remove the test tube 3 after three minutes and cool in a cold water bath; Remove the test tube 4 after five minutes and cool in a cold water bath.
Prepare your reaction vial by mixing 2 ml of amylose solution with 2 ml of pH 7 buffer solution. Add 1 ml of your heated concentrated amylase (Team A will use unheated concentrated amylase), note the time, cap the vial, and shake it. Test samples at one minute intervals for the presence of starch. Record the time required for each reaction.
What effect has heating had on amylase activity? How can this effect be explained?
Summary
You now have investigated the basic facts of enzyme action. Many of the principles which you have learned can be applied to understanding the metabolism of a living cell; however, cells are vastly more complex than the isolated enzyme-substrate systems which you have dealt with. In living cell each chemical process usually consists of many separate steps, each regulated by one or more enzymes, and each dependent upon other chemical processes for its successful completion.