ENZYMES LEARNING OBJECTIVES: After completing this laboratory exercise, you should be able to do the following: 1.

Describe enzyme specificity in terms of the induced-fit theory of enzyme action. 2. Understand how an enzyme speeds up a reaction. 3. Discuss the effects of temperature, enzyme concentration, substrate concentration, pH, and inhibitors on enzyme action. 4. Explain why enzymes are necessary for efficient cell metabolism. 5. Understand the concept of optimal conditions and how it relates to enzyme activity.

INTRODUCTION One of the criteria used to define life is the ability to carry out metabolism or chemical reactions. However, these reactions happen very slowly at normal body temperatures -much too slowly in fact to support life. To increase the speed of these reactions to a point that permits life, organisms use enzymes as biological catalysts. Without enzymes, most biochemical reactions would take place at a rate far too slow to keep pace with the metabolic needs and other life functions of organisms. A catalyst speeds or promotes a reaction without being consumed in the reaction itself. It will not make something occur that is impossible, but the catalyst "pushes" the reaction along by lowering the amount of energy necessary for the reaction to occur -the so-called activation energy .Think of a chemical reaction as a road, and the activation energy as a large hill in that road. The enzyme, a catalyst, reduces the size of the hill, allowing you to "drive" the road using less "gas". For an enzyme to promote a reaction, it must "fit" the molecule or substrate that it is to operate on. The vast majority of enzymes are proteins, therefore, each enzyme consists of a specific sequence of amino acids. Weak hydrogen bonds that form between some of the amino acids help determine the three dimensional shape of the enzyme, and it is this shape that allows the enzyme to fit onto a specific substrate molecule. The specific site which is important in enzyme specificity is called the active site of the enzyme. The shape of the enzyme often changes when the enzyme and substrate interact to produce an induced fit involving an enzyme-substrate complex. The interaction of the enzyme and substrate molecules lowers the activation energy. This may occur by bringing substrates closer together in a position that better favors a reaction, or by altering or straining the covalent bonds of the substrate molecules thus allowing their breakage and reorganization. Although cells contain many enzymes, each kind of enzyme has a precise structure and function, and each enzyme catalyzes a specific reaction. If an enzyme is absent or defective in some way, the reaction will not occur, and the product of that reaction will not be produced. This enzyme defect is the cause of the

a molecule of water is also split.g. Certain genetic defects are correlated with inherited enzyme deficiencies. They may work by binding to the enzyme's active site (steric inhibition) in place of. All chemical reactions that occur in the body require enzymes. Different enzymes have different optimal conditions. an enzyme found in human saliva and in other organisms. or they may bind to another site on the enzyme (allosteric inhibition). 3. into its component sugar units by hydrolysis. These are typically the vitamins and minerals we obtain from a well-balanced diet. This breaking of a chemical bond with the insertion of the ions of a water molecule is . and of the condition phenylketonuria (toxic build-up of certain metabolic by. Starches are stored by plants (eg. Freezing and refrigeration stop or slow down enzymatic activity -thus preventing or retarding decay. Starch is broken down by amylase. 6. Enzymes are used in laundry detergents. APPLICATIONS 1. rice) and by animals (in the liver as glycogen) for use as an energy source. 7. or in acidic conditions (digestive enzymes in your stomach). and the OH. The enzymes in many metabolic pathways are inhibited (and hence the reaction controlled) by the end product of the reaction they catalyze (feedback inhibition).. When the bonds between adjacent glucose units are broken.and H+ ions bind to the exposed ends of the broken starch polymer. Many drugs and toxic agents act by inhibiting enzymes. Conditions in the immediate environment affect the shape of the enzyme by modifying the active site and precise fit of the enzyme and it's substrate. albinism (lack of melanin production). 6. Starch is a polysaccharide composed of glucose molecules linked together by glycosidic bonds. 5.products). changing it's shape and hence the fit of the active site. and thus blocking. 4. Effect of Amylase Activity on Starch Hydolysis. potatoes. Enzyme inhibitors prevent an enzyme from catalyzing a reaction or slow down the enzyme’s reaction rate.hereditary condition. Enzymes are used to help breakdown waste in septic tanks. e. Enzymes frequently need organic molecules and metals to accomplish their function. nerve gas irreversibly binds to the enzyme acetylcholinesterase which plays a critical role in nerve impulse transmission. Enzymes are used to tenderize meat. 2.. Some work best at very high temperatures (those of thermophillic bacteria). the real substrate (their shape is very similar to that of the substrate).

Step 1 The Basic Reaction Method: Fill a 100ml. you can follow the rate of hydrolysis of starch by amylase by using iodine as an indicator for the presence of starch. Time for complete hydrolysis of starch by amylase at room temperature = min. 5 min. and carbon dioxide. 3 min. Glucose is what cells use in cellular respiration to produce energy (ATP). hydrolysis is incomplete. 1 min. and then test the starch/amylase mixture for starch hydolysis. is obtained. water. 10 min. 11 min. Note the color. Now with another clean pipette. Wash out the chem plate in readiness for the next experiment. 9 min. If you test a sample at various intervals after adding the enzyme. the solution will not turn blue-black but will instead turn a variety of colors depending on how much starch remains. If the solution turns blue/black. Starch is hydrolyzed by amylases that cleave the starch molecule into smaller and smaller subunits until maltose. Test the starch/amylase mixture in the remaining depressions at the appropriate time intervals.termed hydrolysis. Note the color. This is the basic dark color indicating the presence of starch. the solution turns a deep blue-black. add 5 ml starch solution to a test tube. Take a temperature reading to estimate room temperature. 7 min. Maltose is subsequently enzymatically converted into individual glucose molecules. Note the time elapsed since you began testing. In the laboratory. 6 min. Add 3 drops of iodine indicator. you will see a graded series of colors from black to deep blue (starch present) to reddish-brown (partial hydrolysis) to yellow-brown. pour about 2Oml of the amylase solution. a disaccharide (2-glucose unit). In another separate small container. If amylase is added to a starch solution. At your desk. Immediately add 3 drops of iodine. place it on a sheet of white paper for better contrast. all starch has now been broken down). Wait until 1 min has elapsed since adding the enzyme. Use a clean pipette to transfer 3 drops of starch solution to the depression marked 0. 8 min. add 2 ml of the enzyme solution to the test-tube with 5 ml starch substrate. 4 min. This will be the substrate for the enzyme Take a chem plate and label a series of depressions for the time intervals you wish to test (0 min. Continue until the starch has been completely hydrolyzed by the amylase (fluid in the depression remains the color of iodine).). beaker about half-full with 1% starch solution from one of the stock solutions made up on the front desk. If you are using the clear plastic chem plate. the color of iodine (complete hydrolysis. Agitate gently. If iodine is added to a starch solution. hydrolysis of the starch will occur -the starch is broken down and when iodine is then added. Room temperature (C) = . 2 min. Pipette out 3 drops of the mixture from the test tube and place in the chem plate depression marked 1. .

water or your fingers. you may proceed. Add 5 ml stock 1% starch suspension to each tube. However. The boiling water must be at a rolling boil. When it matches the experimental temperature (in about five minutes). Use separate pipettes for each temperature to avoid contamination amongst the experimental set-ups.Part 2. If no hydrolysis occurs within 30min. Increasing temperatures therefore increase the number of collisions between enzyme and substrate. Without moving the tubes from their locations. Heat increases the rate of movement of molecules. Use the supplied tongs/holders for manipulating the test-tubes in the hot-water and be careful not to spill or splash the hot liquids! NOTE: Before you add the enzyme to the substrate. terminate the experiment. enzymes generally have an optimal range of temperatures. Temperatures above the optimum for a specific enzyme disrupt the bonds between certain parts of the enzyme molecule. Record the time till complete hydrolysis. The Effect of Temperature on the Rate of Hydrolysis Temperature has a profound effect on enzyme kinetics. After 1 min. start testing for the presence of starch in all 4 test tubes using a chem plate and iodine as you did for the previous experiment. Place C in the ice-bath. H. you must let the test tube warm or cool to the experimental temperature. Even if complete hydrolysis does not occur. H in the palm of your hand with your fingers wrapped around it. Method: Take four test tubes and label them C. note any changes in color compared to the initial example you see when testing with the iodine. Temperature(C) Time to Complete Hydrolysis (min) . put W into a warm water bath (beaker on hot plate) at about 5OºC. and are expected to increase reaction rate. Tube C (Cold) H (Hand-held) W (Warm) B (Boiling) . Wand B. Use a thermometer to assess the exact temperature of all the experimental set-ups. and B in boiling water in another beaker on a hot plate. Gently swirl the tubes. and higher temperatures do not always further accelerate reaction rate. thus distorting it's shape and impeding its function -the enzyme is said to be denatured. add 2 ml of amylase to each test tube. Make sure the liquid-filled parts of the tubes are completely covered by ice.

start removing and testing the solution with iodine as before. Record the times to complete hydrolysis. Enzyme Conc. B. 4 ml to tube B. but only up to a certain point. 1 %. Method: Take four test tubes and label them A.Part 3: The Effect of Enzyme Concentration on Reaction Rate The greater the amount of enzyme. the greater the number of active sites available to the substrate. . In a living organism. Once all the enzyme active sites are occupied. but note any color variations that you see. cells can control the rate of reactions by altering the concentrations of enzymes. but the substrate concentration is increased. 2%. Tube A B C .(ml) Time to Complete Hydrolysis (min) Part 4: The Effect of Substrate Concentration on Reaction Rate If the enzyme concentration is kept constant. place the tubes in a test tube rack. and 4% w/v respectively and place it in a test tube rack. Method: Take three test tubes and label them A. and C. and 6 ml to tube C. the reaction is at its maximum velocity (V max) and any further increase in substrate will not increase the speed of the reaction. initially the reaction rate will increase. B. Now add 2 ml of enzyme solution to each of the four tubes. C and D. If the concentration of a substrate increases dramatically. Add 2 ml of enzyme solution to tube A. If no hydrolysis occurs within 30min. Gently swirl the tubes. Gently swirl the tubes and after 30s start testing samples of the mixtures with iodine in the chem plate as before. Fill each tube with 5 ml 1% starch suspension. After 1 min. terminate the experiment. and thus the faster the reaction rate.5%. Fill each tube with starch suspension equivalent to 0. the cells can respond by making more enzyme.

Fill A with 5 ml water that has been adjusted to pH 5 and place in rack. attracting or repelling each other. After 1 min. Method: Take three test tubes and label them A. The amino acids have different chemical groups attached to them. To each of these tubes. alkaline or basic solutions have a high pH). C and D.Tube A B C D Substrate Conc. and the subsequent shape of the enzyme is essential for its proper functioning. the enzyme molecule loses its shape. Part 5: The Effect of pH on Starch Hydrolysis by Amylase Most enzymes are proteins comprised of chains of linked amino acids. Swirl gently. and consequently its ability to catalyze reactions.4 ml of enzyme solution to each tube and swirl again.(%) Time to Complete Hydrolysis (min) . Some of these molecules interact. tube C with water adjusted to pH 7. start measuring the hydrolysis of starch with the iodine indicator as described before. If the pH is too low or too high. Each enzyme has a pH at which it is most perfectly shaped and thus functions best (optimal pH). The interactions between the chemical groups on the amino acid chain can however be affected strongly by pH (a measure of the concentration of hydrogen ions in solution. acid solutions have a low pH. Similarly. Tube A B C D . fill tube B with water adjusted to pH 6. Add 0. add 1 ml of 1% stock starch suspension. B. and tube D with water adjusted to pH 9. pH Time to Complete Hydrolysis .

Remember to number your graphs. or pH. for each of your data sets. 4. explain the patterns you see in the graphs in terms of molecule movement and how enzymes affect chemical reactions. The graphs will be described in the results section. Enzyme Conc. QUESTIONS 1. each on a separate page. illustrate the data you have collected for each of the experimental set-ups. Write up the laboratory data in the context of a formal report. In the discussion section. or do different enzymes have different optimal conditions? Explain your answer. In other words. . What determines the specificity of an enzyme? Do all enzymes work well at one temperature/pH etc. The horizontal (independent) axis should be labeled with the experimental factor you tested. (ml).GRAPHING THE DATA: Using graph paper. (%). When formulating the hypothesis section. These graphs will be a significant component of your written report. Would amlyase hydrolyse cellulose as efficiently as starch? Why or why not? 3. Hand in both the report and the answers to the questions in the lab exercise for grading next week. Temperature ( C). Substrate Conc. and place legends beneath them. it is advisable to state separate hypotheses for each of the factors you examined. Use a separate graph. Compare what your data illustrate with the theories described in the lab book or text. 2. What temperature does your graph indicate was optimal for amylase? Is this to be expected given where amylase is found? Cellulose is a polysaccharide made of glucose subunits just like starch. The vertical or Y (dependent) axis will be labeled Time to Complete Hydrolysis (min). the data you present should be assessed in terms of what they illustrate about enzyme dynamics..

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