Factors Affecting Enzyme Activity. The rate at which an enzyme works is
influenced by several factors including temperature and pH. Enzymes are most
effective ...
Hillsborough Community College - Ybor City Campus 1025C Laboratory Exercise 3: Characteristics of Enzymes Introduction What are Enzymes? Enzymes are very large and complex proteins that that function as catalysts. They are synthesized by the cell to perform very specific functions. Each type of enzyme has a very specific shape (conformation) which provides its specific function. The shape of an enzyme molecule is determined and maintained by many weak intermolecular interactions between many different parts of the molecule. A catalyst is a material that donates energy to reactants in order to reduce their activation energy. In other words, they allow reactions to occur with less energy input (i.e. more quickly or at lower temperatures). The very specific shape of enzymes allows them to “fit” into a reaction and reduce the energy necessary for the reaction to occur. If their shape is changed, their ability to function as a catalyst is reduced or eliminated. As biological catalysts, enzymes are important because they speed up the rate of the reaction they catalyze that would otherwise be too slow to support life. Factors Affecting Enzyme Activity The rate at which an enzyme works is influenced by several factors including temperature and pH. Enzymes are most effective as catalysts under optimum physical and chemical conditions; as conditions change away from optimum, enzyme activity decreases (Figure 1.). Changes in various environmental factors, such as temperature or pH, may affect proteins by altering their shape; with loss of shape comes loss of function causing the protein to become denatured. Denaturation is often a reversible process.
Figure 1. Effects of Variable Temperature and pH on Enzyme Activity
The Effects of Temperature on Enzyme Activity Under ordinary conditions, at any given temperature, molecules possess a range of kinetic energy (Ek). A portion of the molecules have sufficiently high kinetic energy to allow them to react (Ea = activation energy). As temperature increases, the proportion of molecules possessing high energy increases as does the proportion possessing activation energy (Ea) (Figure 2.) As the proportion of molecules possessing Ea increases, the rate at which a reaction takes place increases.
Molecules
Kinetic Energy (EK)
Ea
Figure 2. Increasing Kinetic Energy (Ek) and Proportion of Molecules with Activation Energy (Ea) with Increasing Temperature (T2 > T1)
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In general, the rates of chemical reactions increase as temperature increases and decrease as temperature decreases as a function of the availability of activation energy. When the temperature increases, more of the molecules have the kinetic energy required to react. When temperature decreases fewer molecules possess that activation energy. Likewise, enzyme-catalyzed reactions also tend to be slower at temperatures below the optimum. They also tend to go faster with increasing temperature but only until a temperature optimum is reached. Above the optimum temperature, the Ek of the enzyme becomes great enough that the weak intermolecular attractions that maintain the shape of proteins are broken and the enzyme molecule is disrupted - the enzyme becomes denatured. Changing the shape of the enzyme results in less efficient binding of the substrate (reactants) resulting in a significant decrease in enzyme activity. Temperatures above 40-50°C (104-122°F) denature many enzymes. The Effects of pH on Enzyme Activity pH is a measure of the acidity or hydrogen ion concentration of a solution. It is measured on a scale of 0-14 with pH values below 7 being acidic, values above 7 being alkaline and a value of 7 being neutral. As the pH changes away from the optimum toward the acidic range of the pH scale, an enzyme tends to gain hydrogen ions from the solution. As the pH changes away from the optimum toward the alkaline range of the pH scale, the enzyme tends to lose hydrogen ions to the solution. In both cases, changes are produced in the weak interactions that maintain the shape of the enzyme molecule resulting in a change in shape that denatures the enzyme and decreases the enzyme activity. The Effects of Surface Area on Enzyme Activity In this exercise, the catalase enzyme is available only at the surface of the potato. As potato pieces get smaller, more surface area is exposed (= more enzyme available to react) and the overall enzyme activity becomes greater. This process works in the opposite manner in your digestive system. Mechanical digestion reduces food particles to very small sizes (powder-like) making a large amount of surface area available for digestive enzymes to act upon.
Figure 3. The Effect of Particle Size on Surface Area
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Name: Hillsborough Community College - Ybor City Campus 1025C Laboratory Exercise 3: Characteristics of Enzymes Catalase is an enzyme that is present in many living cells to help convert hydrogen peroxide (H2 O2) to water and oxygen gas (H2O + O2 ). Hydrogen peroxide is a product of lipid metabolism. It is a strong oxidizer, causing free radical formation which can be very toxic and damaging to cells. It is important for cells to have enzymes such as catalase to protect themselves against free radical formation by breaking hydrogen peroxide down into safe and useful substances. As the catalase reaction occurs in a test tube, effervescence (bubbling) develops that is easily observable as oxygen gas is formed. The general equation for this reaction is: 2 H2O2 + catalase
2 H2O + O2 + catalase.
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For the experiments in Part 1 and Part 2, record your observations of results in this manner: No effervescence = 0 Minor effervescence = + Moderate effervescence = ++ Good effervescence = +++ Very good effervescence = ++++
PART 1: The Effect of Temperature and Surface Area on Catalase Activity In Part 1 of this exercise, you will be observing the effects of two variables on the activity of catalase. 1. Temperature (cooked vs. raw potato = high vs. low temperature) 2. Surface area (large piece vs. small pieces = low vs. high surface area) You will need to identify the experimental control in this procedure. Procedure Work in groups of two. •
Label 5 test tubes 1-5 and mark each with a line 4 cm from the bottom.
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Tube 1: Add a small amount of sand (just enough to cover the tube’s bottom).
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Tube 2: Add a small piece of raw potato.
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Tube 3: Add small piece of raw potato about the same size as the piece in tube 2, but chop the piece of potato into smaller pieces.
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Tube 4: Add a small piece of cooked potato.
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Tube 5: Add a small piece of cooked potato about the same size as in tube 4, but chop the potato into smaller pieces.
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Keep the quantity of potato in tubes 2 and 3 approximately equal to the quantity of potato in tubes 4 and 5.
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Add hydrogen peroxide to the 4 cm mark on each tube.
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Observe and record your results in Table 1 and provide some possible explanations for the differences you observe in terms of the two variables (temperature and surface area) that are being tested.
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Table 1 Catalase Activity of Potato with Variable Temperature and Surface Area TUBE
Temperature
Surface area
RESULTS
1
2
3
4
5
What is the experimental control in this procedure? 21
EXPLANATION
PART 2: The Effect of pH on Catalase Activity In Part 2 of this exercise, you will observe the effect of pH on the activity of catalase. 1. HCl = low pH (acidic) 2. NaOH = high pH (alkaline) 3. You will need to identify the experimental control in this procedure. Procedure •
Label three test tubes 1-3 and mark each with lines at 2 cm and 7 cm from the bottom.
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Add equal amounts of chopped raw potato to the three tubes.
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Tube 1: Add water to the 2 cm mark.
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Tube 2: Add 0.1M hydrochloric acid (HCl) to the 2 cm mark.
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Tube 3: Add 0.1M sodium hydroxide (NaOH) to the 2 cm mark.
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Wait 5 minutes.
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Add hydrogen peroxide to the 7 cm mark of each tube.
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Use pH paper to determine the pH of the solution in each tube and record these values in Table 2.
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Observe and record your results in Table 2 and provide some possible explanations for the differences you observe in terms of the variable (pH) that is being tested. Table 2 Catalase Activity of Potato with Variable pH
TUBE
pH
RESULTS
EXPLANATION
1
2
3
What is the experimental control in this procedure?
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Hillsborough Community College - Ybor City Campus 1025C Laboratory Exercise 3: Characteristics of Enzymes Review Questions 1.
In Part I of this exercise, two variables (conditions) are being tested to determine their effect on the observable reaction of catalase with H2O2. What are these two variables and why do you think each might have an effect on catalase activity?
2.
What is the control for the experiment in Part I of this exercise? Why is it necessary?
3.
What do you suppose your observations might be if you prepared a sixth tube just like the third tube (chopped, raw potato) but placed it in a refrigerator for an hour before you added the H2O2?
4.
In Part II of this exercise, what variable is being tested for its effect on the activity of catalase?
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5.
Explain briefly why this variable might be able to alter catalase activity.
6.
Explain how the human digestive process affects the variables seen in the experiments in Part I and Part II of this exercise.
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