Cata
Potato catalase: a study of enzyme action
Introduction
Thousands of chemical reactions take place within living cells of organisms. Many of these reactions can also be carried out in the laboratory, but outside the cell, they are often much slower. To speed up these reactions so they would take place as fast as they do in cells would require temperatures or pHs that are inconsistent with life. How then is it possible for reactions to occur in cells at rates fast enough to meet the needs of the body? The answer is that cells produce special proteins known as enzymes that catalyze biological reactions, markedly increasing their rates.
The primary species acted on by an enzyme is called the substrate of that enzyme. Most enzymes are named after the substrate on which they act by simply adding –ase to the root name of the substrate. A lipase then, would be an enzyme that acts on lipids, sucrase on sucrose, and so forth. Many enzymes, such as chymotrypsin, trypsin and lysozyme, have older names that do not end in –ase.
Some enzymes owe their specific reactivity only to their specific protein structure. Others, however, are conjugated proteins that require the presence of a unique nonprotein unit to become an active enzyme. The protein portion of a conjugated enzyme is known as an apoenzyme, and its nonprotein portion is termed a cofactor. There are two kinds of cofactors. If the cofactor is an organic unit, it is commonly called a coenzyme. If the cofactor is a metal ion, it is called a metal-ion activator. Some enzymes require both types of cofactors. Na+, K+, Mg2+, Ca2+, Mn2+, Co2+, and Zn2+ are examples of metal-ion activators. Many trace metal ions found in the body are important in enzyme reactions. Some vitamins or their derivatives are coenzymes. Since vitamins and metal ions (often referred to as minerals) are essential for proper enzyme function, it is easy to understand why they are essential components of the diet.
Uses of Enzymes
Enzymes serve many critical functions in the body, catalyzing nearly every reaction that takes place. In recent years, researchers have learned to use enzymes to serve our needs in various ways. Enzymes can be powerful diagnostic tools in medicine, and it is not difficult to measure quickly and accurately the level of a specific enzyme in blood serum or urine. Normally, enzymes appear in blood serum or other extracellular fluids at very low concentrations, but certain disease conditions can markedly increase the level of one or more of them. If disease or injury damages the cell membrane, the enzymes will flow out into the extracellular fluid and eventually enter the bloodstream, where they can be detected easily. Of course, the use of enzymes is not restricted to health care. Commercially, proteolytic enzymes, such as papain, are used to tenderize meat. They catalyze the hydrolysis of connective tissue, reducing the toughness of meat. Food manufacturers use enzymes to partially digest food for infants and others with digestive problems. Certain proteolytic enzymes are used to remove cataracts.
Enzyme Action
Enzymes are efficient biological catalysts. They increase the rate of a reaction by lowering its activation energy, the energy barrier that must be crossed over as reactants are converted to products. For example, the decomposition of hydrogen peroxide, H2O2, to form oxygen and water has an activation energy in the absence of catalyst of 18 kcal per mole of H2O2. This same reaction occurs in cells, but in the presence of an enzyme called catalase. Catalase reduces the activation energy to around 7 kcal per mole of H2O2, a substantial reduction, which in turn allows the reaction to take place about 100 million times faster. The effect of catalase on the activation energy of this reaction is shown in Figure 1.
The efficiency of an enzyme is given by its turnover number, the number of substrate molecules that one enzyme can convert to product in one minute. At body temperature, the turnover number for catalase is 5.6 million. This means that one catalse unit can pick up and decompose 5.6 million H2O2 molecules each minute.
Figure 1. Effect of an enzyme on activation energy
An enzyme catalyzes a reaction by changing the pathway that leads from reactants to products; that is, it changes what is called the “mechanism” of the reaction. It does this by providing a special surface for the substrate molecule (or molecules) to fit on so that bonds can be broken and formed much more easily. The special surface is called the active site of the enzyme. The active site is usually a small crevice or cavity formed in the tertiary structure of the protein. The active site on an enzyme has a specific geometry or shape that will accommodate only specific substrate molecules that can fit into it. The substrate molecule, therefore, must have a structure that complements the structure of the active site, so that the two fit together like a key into a lock. This model of enzyme substrate complex formation is called the lock-and-key theory of enzyme action. The enzyme is the key that “unlocks” or changes the substrate molecule. The cofactor that is required for the enzyme to function either may be part of the active site or may bond elsewhere to the surface of the enzyme and assist in a necessary but less direct way.
Let us examine a step-by-step sequence of events that would be typical for an enzyme-catalyzed reaction that breaks a substrate molecule into two parts. The mechanism of the reaction can be described by three equations representing the three steps of the process. Let E symbolize the enzyme and S the substrate. P is the product of the reaction. Note that each step is reversible.
If the three steps are added together, the overall reaction is simply the conversion of substrate to product (S ® P). Since the enzyme catalyzes the reaction, “enzyme” is written over the arrow in the equation:
substrate ¾¾¾¾¾® product
Factors that Influence Enzyme Catalyzed Reactions
1. Enzyme Concentration
As the concentration of an enzyme increases, the rate at which the substrate is changed also increases. If one enzyme molecule can transform a million substrate molecules each minute, then two enzyme molecules will handle twice that number of substrate molecules in the same time. The substrate will be consumed twice as fast, which doubles the rate of reaction. The way the rate of an enzyme-catalyzed reaction is affected by the concentration of enzyme is shown in Figure 2. As long as sufficient substrate is present, the reaction is proportional to the enzyme concentration, i.e. if you double the enzyme concentration you will double the rate of reaction.
Figure 2. Effect of enzyme concentration on the rate of reaction
2. Substrate Concentration
For any given enzyme, there is some maximum number of substrate molecules that it can transform or turn over each minute. If this number is very large, the reaction will have a high maximum rate. Now let us consider a single enzyme molecule in a solution of substrate molecules. If the concen-tration of the substrate is very low, the number of substrate molecules that can be “caught” by the enzyme each minute will also be small. It will be lower than the number of sub- strate molecules the enzyme is capable of handling during that time. The rate of the reaction, as measured by the number of substrate molecules changed each minute, will also be low, lower than the maximum rate. If the concentration of sub- strate is increased, the rate of the reaction will also increase until a point is reached where the enzyme is working as fast as it can. At this point, the enzyme is said to be saturated, and further increases in the concentration of the substrate will not increase the rate of reaction. The enzyme can work no faster. The effect of substrate concentration on the rate of an enzyme catalyzed reaction is shown in Figure 3.
Figure 3. Effect of substrate concentration on the rate of reaction
3. Temperature
The rate of an enzyme catalyzed reaction increases as the temperature of the medium increases, but only up to a point. For every enzyme, there is one temperature at which the reaction rate will be at a maximum, and that temperature is called the optimum temperature for that enzyme. The rate of the reaction at temperatures above or below the optimum temperature will be slower. The optimum temperature for most of the enzymes in the body is approximately 37°C, normal body temperature. Figure 4 shows the effect of temperature on an enzyme-catalyzed reaction.
At temperatures above 60°C, many enzymes will denature, destroying their secondary and tertiary structures, which in turn destroys the active sites. At these temperatures, the activity of the enzyme is reduced to zero. When milk is pasteurized, the high temperatures used in these processes destroy not only bacteria, but also enzymes. Low temperatures do not denature enzymes. Tissue samples, sperm, and other biological materials can be stored at sub- freezing temperatures for years without markedly reducing the catalyzing power of the enzymes present in the samples.
Figure 4. The effect of temperature on an enzyme catalyzed reaction
4. pH
Just as there is an optimum temperature for enzyme activity, there is an optimum pH at which an enzyme’s activity is greatest. At pHs above and below the optimum pH, the activity of the enzyme is reduced and reaction rates are slower, as shown in Figure 5. The optimum pH for pepsin, a proteolytic enzyme in the stomach, is around 2, close to that of the acidic environment of the stomach. Trypsin, another proteolytic enzyme, has an optimum pH around 8, close to that of the upper intestinal tract where it is found. If trypsin were placed in the highly acidic environment of the stomach, it would likely denature and lose its catalytic activity.
Figure 5. Effect of pH on the rate of an enzyme catalyzed reaction
5. Cofactor Concentration
Cofactors are essential parts of active enzymes. If the proper cofactor is absent, an enzyme will have little or no activity. For this reason, cofactors must be present in sufficient concentration to activate enzyme molecules. If they are not, then only a fraction of the enzyme concentration will be effective in catalyzing reactions.
6. Enzyme Inhibitors
Enzyme inhibitors are substances that slow or completely stop enzyme-catalyzed reactions. This inhibition may be reversible or irreversible. Natural enzyme inhibitors occur in living systems to control the rates of specific reactions, thus preventing the formation of more reaction product than is needed. Enzyme inhibitors include a number of poisons, insecticides, herbicides and antibiotics. Some well-known enzyme inhibiting poisons include the cyanide ion, CN-, arsenate ion, AsO43-, copper (II) ion, Cu2+, and heavy metal ions like Hg22+, Hg2+, Ag+, and Pb2+.
Objectives
1. To measure the rate of an enzyme catalyzed reaction.
2. To determine the optimum temperature and pH of the reaction catalyzed by potato catalase.
3. To determine the effects denaturation and inhibitors have on the rate of the catalase reaction.
Procedure
We will study the decomposition of hydrogen peroxide (H2O2) by the enzyme catalase obtained from potato. Hydrogen peroxide is produced in many plant and animal tissues and it would be toxic if allowed to build up. The equation for the decomposition of hydrogen peroxide by catalase is:
2
2 H2O2 (aq) ¾¾¾¾® 2 H2O (l) + O2 (g)
The rate of decomposition will be determined by measuring the amount of O2(g) produced. The apparatus shown in Figure 6 will be used to measure the volume of oxygen produced as the hydrogen peroxide decomposition takes place. The evolved oxygen will displace water from the inverted 10 mL graduated cylinder.
Figure 6
Preparation of apparatus
1. Fill a pneumatic trough two-thirds full of tap water. Then completely fill a 10 mL graduated cylinder with tap water. Place your finger securely over the mouth of the cylinder, invert it, and immerse the sealed mouth beneath the water level in the pneumatic trough. Remove your finger and secure the inverted graduate to a ring stand using a clamp.
2. Using a second clamp, attach a 15-cm sidearm test tube to the ring stand and lower it into a 250-mL beaker. Fit the sidearm test tube with a tight fitting stopper.
3. Fit a length of rubber tubing onto the sidearm test tube. It should be long enough to reach to the graduated cylinder. A short section of bent glass tubing on one end of the rubber tubing will help direct the flow of oxygen gas into the inverted graduate.
4. Obtain two more 10 mL graduated cylinders. One will be used solely for measuring catalase. The second will be used solely for measuring hydrogen peroxide. Each must be rinsed three times with deionized water and once with the solution to be measured before use.
Preparation of catalase (Catalase may already be prepared for you. Check with your instructor.)
1. Obtain about 30 g of peeled raw potato cut into small pieces.
2. Mash the raw potato using a mortar and pestle until it forms a paste.
3. Place the paste into a 100 mL beaker which contains 30 mL of deionized water and stir for 10 minutes.
4. Filter the solution through a cheese cloth and into an Erlenmeyer flask.
5. After 5 minutes the solution is ready to use.
A. Effect of Temperature on Enzyme Activity
The catalyzed rate of decomposition of hydrogen peroxide at four temperatures will be measured and compared. Water will be placed into the 250 mL beaker at four different temperatures as follows: 1) an ice bath, 2) water at room temperature, 3) water at about 37°C, and 4) water at about 60°C.