Exercise 6: Enzymes________________________________________________________________________

OBJECTIVES: Learn about the function of enzymes. Understand and be able to predict how temperature affects enzyme activity.

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INTRODUCTION:

Biological processes, including growth, reproduction and metabolism, require a constant supply of energy. The production of energy is accomplished through thousands of chemical reactions that occur in cells and are regulated by protein catalysts called enzymes. Our everyday lives are dependent on these proteins and in some instances the absence of a particular enzyme can cause serious illness or even death.

The use of enzymes in our daily lives has significantly improved our standard of living. For instance, proteases and amylases, which are produced by the body to break down protein and starch, respectively, are also used in commercial baking. In medicine, enzymes are important agents in the treatment of heart attacks (streptokinase dissolves blood clots in arteries of heart walls) and cancer (asparaginase is used for acute lymphocytic leukemia in children). Without enzymes, life would not be possible. Food would not be converted to energy and our bodies would not be able to replace old, damaged tissues with new, healthy ones. Cellular waste products would not be disposed of, and ultimately, all cellular activity (metabolism, reproduction, growth) would cease.

Enzymes increase the rate of reactions by lowering the activation energy (energy needed to start a reaction) required for chemical reactions to occur (Fig. 1). Without these catalysts, all metabolic processes would take far too long to sustain life.

Figure 1. Enzymes lower the activation energy needed for chemical reactions

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Most enzymes are proteins with three-dimensional shapes determined by their amino acid sequences. When a substrate (reactant) molecule binds to the highly specific active site of an enzyme, an enzyme-substrate (ES) complex is formed (Fig. 2). The ES complex modifies the substrate’s chemical bonds and initiates a series of chemical reactions resulting in the formation of a product. During the reaction, the enzyme is not changed or consumed and thus it is reusable. As products are generated they are released from the enzyme’s active site allowing other substrate molecules to bind.

Figure 2. Substrate binding at the active site of an enzyme. As the substrates (blue and red) bind to the active site (1), the enzyme-substrate (ES) complex forms (2), and the products are released (3).

The active site determines the specificity of every enzyme. Specificity can result from the charge, shape, and/or hydrophobic/hydrophilic characteristics of the enzyme and substrate molecules. Only reactants that match the shape of the active site can bind to the enzyme - the “lock and key model” where the substrate (the key) fits into the active site (the lock) of an enzyme. However, the interaction between a substrate and the enzyme’s active site is not static. When a substrate binds to the enzyme, the active site is reshaped by the interactions of the enzyme’s amino acid side chains with the substrate molecule. This protein remodeling enhances the overall binding of the reactant to the active site, increasing catalytic action. The ability of enzymes to mould their shape to enhance the fit of substrate molecules is known as the “induced fit model.”

There are thousands of different enzyme types, each with a specific set of conditions at which it works best, i.e., its optimal conditions. An enzyme’s optimal conditions often reflect the environment(s) of the organism(s) in which it is found. For instance, the optimum temperature for enzymes present in Thermophilus aquaticus, an extremophilic bacterium that inhabits hot springs, is about 70ºC. In contrast, peroxidase, an enzyme present at high concentrations in turnips, horseradish roots and potatoes, works best at temperatures around 45ºC. Enzymatic activity is affected by multiple factors, including pH, substrate concentration, salt concentration, as well as the presence

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Fig. 5.6 How enzymes workFig. 5.6 How enzymes workFig. 5.6 How enzymes work

of inhibitors, activators and cofactors. In this lab, you will examine the effect of temperature on enzymatic reactions.

Temperature affects the rate at which substrate and enzyme molecules collide. At temperatures greater than the optimal, the active site denatures (i.e. changes shape), decreasing or preventing substrate binding. Consequently, product formation is either reduced or completely arrested. At low temperatures the movement of molecules decreases, resulting in less contact between enzymes and substrates, which slows down the frequency and rate of reaction, and ultimately diminishes product formation. The objective of the current lab is (1) to examine how variations in temperature affect the activity of the enzyme amylase, and (2) to determine the optimal temperature for amylase from two different sources (fungal and human).

Amylase catabolizes starch polymers (a storage polysaccharide) into smaller subunits (monomers = saccharides) including maltriose, maltose and short oligosaccharides comprised of 2-20 monosaccharide units (Fig. 3). Most organisms use these saccharides as a food source and to store energy. Both starch and amylase are important commercially in the production of syrups and other food products, as well as for fermentation and brewing processes.

Figure 3. Starch digestion by amylase________________________________________________________________________

TASK 1 - Effect of Temperature on Amylase Activity

The objective of this exercise is to determine the optimal temperatures for fungal (Aspergillus oryzae) and human amylases. In addition, you will examine the effect of temperature on the ability of amylase to break down starch to maltose. You will monitor starch catalysis visually using the Iodine test, which turns from yellow to blue-black in the presence of starch (for further details see Exercise 4).

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Questions:

a. What do you expect to happen to starch breakdown with increasing temperature?

Write your hypotheses (Ho and Ha) below.

b. What criteria will you use to determine that starch has been catabolized?

c. Do you expect the fungal and human amylases to have the same optimal temperatures? Explain.

Write your hypotheses (Ho and Ha) below.

Before beginning the experiment, predict what you expect to occur for each of the four temperature treatments. Record your predictions in Table 1.

Table 1:Temperature

(oC)Fungal Amylase Expected results

Human Amylase Expected results

Reason(s) for Expected Outcome

0

40

60

95

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Important Notes: At the end of the experiment, your group should have 2 data sets, one for

each type of amylase (fungal and human). To determine the optimal temperatures for both enzymes, you will need to

generate a class data set by combining your group’s results with the data collected by the other groups in your class.

Procedure:

I. Experimental Setup: 1. Set up 2 spot plates as shown in Figure 4. Place a napkin under the spot plates

and across the top write Temperature (0°, 40°, 60°, 95° Celsius) and on the side write Time (0, 2, 4, 6, 8, 10 min).

Figure 4. Spot plate setup

2. Obtain 4 test tubes and label each with a different temperature (0°, 40°, 60°, 95° Celsius), your enzyme source (H - human and F - fungal) and your group number.

3. Obtain another 4 test tubes and label these identically to the 4 in step 2 except add an additional letter S (for starch solution).

4. Add 5mL of 1% starch solution into each of the 4 test tubes labeled S.

II. Effect of Temperature on Amylase Activity

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5. Add 1mL of amylase into each of the test tubes that do not contain starch.a. If you have been assigned Human amylase (H), collect saliva from

each member of your group into a weighing boat. If you ate before coming to lab make sure to rinse out your mouth with water prior to saliva collection.

i. Transfer 0.5mL saliva into each test tube.ii. Dilute your saliva by adding 0.5mL of distilled water into each

test tube containing the saliva.b. If you have been assigned Fungal amylase (F), add 1mL of fungal

amylase solution to each tube.

6. Place all 8 test tubes including the 4 containing starch as well as the 4 with the amylase into their respective temperatures. ***PRIOR TO PUTTING THE TEST TUBES INTO THE WATER BATHS MAKE SURE THE TEMPERATURE FOR THE WARM WATER BATHS ARE STABLE!

a. Place tubes labeled 0ºC into the ice bath, b. 40ºC into the 40ºC water bath, c. 60ºC into the 60ºC water bath, d. 95ºC into the 95ºC water bath.

7. Allow all tubes to equilibrate for 5 min in their respective temperatures.

8. Meanwhile, add 2 drops of iodine into each well on the spot plates.

9. At the end of the equilibration process, without removing the tubes from their water baths, transfer a few drops of the starch solution from each temperature treatment to the first row of the spot plate corresponding to time 0. Make sure to use a separate transfer pipette for each temperature treatment.  Label each of your transfer pipettes with the correct temperature so that they can be reused for each time interval

10. Within each temperature treatment, pour the starch solutions from the 4 tubes into the tubes containing amylase. Set your timer for 2 min at the moment of amylase addition.

11. After 2 min, use the correct transfer pipette for each temperature to remove a few drops of the starch-amylase mixture from each tube. Place 2-3 drops of the mixture in the second row (time = 2 min) on your spot plate under the correct temperature. Note the color changes and record your observations in Table 2 (human amylase) or 3 (fungal amylase). Assign a quantitative value in Tables 2 and 3 for each solution based on Figure 6 (below) immediately after transferring the solutions to the spot plate.

12. Use the color-coding scheme below (Fig. 6) to convert your results (qualitative data) into quantitative (numerical) data. In the column next to your color data, record the corresponding number.

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STARCH HYDROLYSIS

1 2 3 4 5

MOST STARCH LEAST

Figure 6. Starch hydrolysis

13. After each additional 2 min, repeat step 10. Make sure that you add the starch-amylase mix to the correct wells for time and temperature.

14. At the end of the 10 min, note the temperature and the time at which 100% hydrolysis occurred (Fig. 6).

Table 2: Human amylase

Temperature (oC)

0 40  60 95

Color # Color # Color # Color #

Tim

e (m

in)

0    

2        

4        

6        

8

10        

Table 3: Fungal amylase

Temperature (oC)

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0 40  60 95

Color # Color # Color # Color #T

ime

(min

)

0    

2        

4        

6        

8

10        

Questions:

a. Which of the variables is (are) the independent variable(s)?

b. Which of the variables is (are) the dependent variable(s)?

c. What are the controls and what do they control for?

d. Based on the color findings, is starch present at time zero? Should starch be present? Why or why not?

e. Did your observed results reflect your predictions (Table 1)? Explain.

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f. What do your results indicate about the optimal temperature for each type of amylase?

g. Explain the relationship between the amount of starch and maltose present during starch hydrolysis.

h. Put your results on the board, and record all the collected data from the class.

i. Calculate the mean and standard deviation for each temperature and time for both enzymes.

j. Using the class data set, create a graph to address the following questions:o How does temperature affect amylase activity?o Is starch catabolism equally efficient across all temperatures?

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o Do fungal and human amylases breakdown starch at the same temperature(s) and rate?

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