TOPIC «& Enzymes OBJECTIVES 1. Deseribe the chemical nature and structural aspects of ‘enzymes 2. Summarize the role of enzymes as chemical regulators sad to deseibe the biological function of the ative sites. INTRODUCTION Proteins have important roles in living organisms. ‘They are amino acid polymers covalently bonded in long chains that are subsequently coiled and folded into complexshapes. Proteins form structural features such as hair, hooves, and tendons, Other proteins, known as enzymes, affect the many biochemical reactions that keep cells alive. Bn- zymes act as catalysts in biochemical reactions. This means tha they speed chemical reactions that would, inthe absence of the enzymes, proceed at very slow rates or perhaps not even at all. Enzymes accomplish this effect by lowering the energy of activation of a biochemical reaction, which isthe energy required to te the reaction, Specific substrates, or reactants, combine with enaymes at their active site(s), which induces biochemical reactions that result in products used by the cell Fig. 6-1). “The activity of a specific enzyme is affected by many factors such as pH, temperature, allosteric in- teractions, and inhibitors (substances that prevent normal action ofthe enzyme). ‘A temperature and/or pH higher than what ace nnocinal for an enzyme will denature (ater the molec- ular shape of) the protein, thereby disabling the en= zyme’s normal activity. A pH lower than normal will 3, Identify the effets of pH{ and temperature on enzyme activity 4, Describe the action and effect ofan inhibitor on enzyme activity have a similar effect. Enzymes are not denatured by lower temperatures, but the kinetic energy is lowered toa point where the rate of biochemical rezctions is greatly reduced, Enzyme activity can bedeereased by inhibitors that bind to the enayme's active site, This prevents sub- strate molecules from binding and no chemical reae- tion oceurs (Fig. 6~2a). Enzyme activity can also be decreased by inhibitors that bind toa site other than theactivesiteof the enzyme. This renders the enzyme inoperative by altering its shape (Fig, 6-26). Some enzymes, termed allosteric enzymes, have smoze than one binding site (the portion ofthe enayme molecule that nozmally comes in contact with the substrate molecule). Such enzymes fequently exhibit ‘dramatic change in activity when one site is oces- pied by a substate o, in some cases, by a molecule similar to the substrate All ofan organism's biochemical xeactions are col Jectively referred to as its metabolism. The reactions involved are produced by a series of enzyme- aediated activities called metabolic pathways. The phenomenon of negative feedback occurs when the concentration of an enzymatically produced product docieases the activity of the enzyme(s) involved, ‘When the product becomes used up, enzyme activityIshibitor Substrate @ FIGURE 6-2 Eneyme Activity: The Effect of Temperature 63 FIGURE 6-1 ‘An enayme speeds upa chemi- cal reaction by lowering is ac- uation energy. A catalyzed reaction proceeds morequickly than an uncetalyzed zeaction Decause the bares of activa: tion is lowered, Active ites not table for rogption of substrates (2) The inkibitor competes withthe normal substrate forthe active ste ofthe enzyme, (b) The inhibitor binds with the enzyme ata site oer than the active sie, altering the shape of the enzyme and thereby inactivating it. resumes once again. Negative feedback is one of a variety of methods of metabolic control in an oxga~ ‘We have stated that enzymes are proteins; how- ever, ithas recently been discovered that certain types of RNA (a nucleic acid) can act as an enzyme, which shows that our knowledge of enzymatic biochemistry is stil growing, The following laboratory exercises demonstrate some of the fascinating properties of enzymes. PRELAB QUESTIONS 1. What are the monomers that compose an enzyme? 2. What is the effect of enzymes on chemical reac- tions? 3, What factors affect enzyme activity? 4. What effect does extreme pH have on normal en- zyme activity? LAB 6.4 ENZYME ACTIVITY: THE EFFECT OF TEMPERATURE MATERIALS REQUIRED. potato extract catechol 5 test tubes test tube rack test tube holder boiling water bath, 100°C ‘warm water bath, 40°C ice bath, 0°C wax pencil ‘small ruler64 Topics Lab 6A. PROCEDURE ‘The enzyme you will be using, catechol oxidase, is found in plant tissues. AS its name implies, catechat onidase oxidizes (removes hydrogen from) the sub- strate catechol, also found in plants, and converts ito ‘benaoquinone, which is brown in color. This reaction explains the darkening of bruised frit and vegeta- les. The injured cells release catechol ané catechol oxidase, which do not contact one another in unbro- een cells The benzoquinone produ, whichis toxic ‘bacteria, helps prevent decay in damaged plant tissue. You will use potato extract as a source of catechol ‘oxidase and a solution of catechol to investigate the various factors that affect enzyme activity (see Fig, 6-3). 1, Use the ruler and wax pencil to accurately mark ‘both 1 cm and 2 om from the dettov of clean text tubes. Number the top end af the test tubes 1, 2,2, sands, 2, Fill tube 1 to the 1 cm mark with distilled water ‘Thistube remains in the test tube rackand wil ater serve as a control 3. Fill tubes 2 through 5 up to the 1 em mark with potato extract. Place tube 2 in the hot water bath, tube 3 in the wacm water bath, (abe 4 in the ice ‘bath, and tube 5 Jn the test tube rack (which re ‘mains at oom temperature). Allow all test tubes to stand for § minutes a their various temperatures. 4. Alter the nubes reach ther termperatures, add cate- ‘hol to tae 2.em mask in all test tubes. CAUTION Catechol is toxic! Use caution ‘when filling test tubes. Wash thoroughly with soap and water if catechol contacts your skin. 5, Watch for any color change in the 5 test tubes dur- ing the next 20 minutes. Draw a table to record color changesatS-minute intervals for 20 minutes. Use the following plus ard minus color intensity scale presented beiow for recording colorintensity ‘Mix the solutions at 5-minute intervals by gently tapping the bottom of the tubes. Be sure to use a test tube holder to handle tube 2 in the hot water bath, Color atensty Seale = =no color change FT venr light colar change +, Slight color change H+ = definite color change HS dark olor CAUTION After recording results, do not cemply test tubes into the drain, Dispose of catechol in the container designated! for catechol disposal POSTLAB QUESTIONS 1. Identify the substrate, the enzyme, and the product involved in this experiment. 2. Was the control lacking the substrate or the en- zyme? 3. In Step 3, why is it necessary 10 lt the test tubes stand for 5 minutes in their various temperatures? 4. Based on your results, what was the optimum tem- perature for catechol oxidase activity? 5. What effect did @ hot water bath have on enzyme activity? Why? 6, Using your results, argue for ot against the follow- ing statement: Enzymes function equally and eff- cently at all temperatures. 7. Why does lemon juice applied to slices of apples or ‘bananas prevent darkening of the fruit?Enzyme Activity The Efect ef Temperature 65 Wialer Potato Potato extaet extract ote for colar in change every nutes for 20 minutes Water Folulo Potato Posie Potato catechol entact+ extracts extrac + extra catechol citecholcetethot catechol « FIGURE Process for66 Topic Lab 6.c LAB 68 ENZYME ACTIVITY: THE EFFECT OF AN INHIBITOR MATERIALS REQUIRED 3 test tubes test tube rack ‘wax pencil small ruler polato extract sodium phosphate solution PTU solution distilled water in dropper bottles catechol PROCEDURE Enayme inhibitors interfere with the fanction of en- zymes by binding with some critical portion of the enzyme molecule. Your objective is to determine ‘which of two solutions, sodiam phosphate (NaPO,) or phenylthiourea (PTU), is an inhibitor of the en- zyme catechol oxidase (see Fig. 6-4). 1, Use the ruler and wax peneil to accurately mark both I cm and 2 cm from the Bottom of 3 clean test tubes, Number the top end of the test tubes 2, 2, and 3 2, Fllall tubes to the 1 cm mark with potato extract. 3. Add five drops of distilled water to tube 1, five drops of sodium phosphate solution to tube 2, and five drops of PTU solution to tube 3 CAUTION PTU is toxic! Flush thoroughly with ranning water ifthe solution contacts skin or clothing. 4, Fill all tubes to the 2cm mark with catechol and mix well 5. Watch for any color changes in the 3 test tubes during the next 20 minutes. Mix the tube contents at Seminute intervals while timing. Record color changes in a table at S-minute intervals using the plus and minus scale in the previous lab. CAUTION After recording results, do not ‘empty PTU solution into the sink drain. Dispose of PTU in the container designated for catechol dis~ posal. POSTLAB QUESTIONS 1. Identify the substrate, the enzyme, the inhibitor, and the product involved in this experiment, 2, What is the purpose of tube 1? 3. What was the effect of an inhibitor on enzyme activity? 4. The enzyme catechol oxidase has 8 copper ion that forms part ofits active site. Knowing this, what do you suggest as the mechanism of action for the imhibitor to affect enzyme activity? LAB 6.C ENZYME ACTIVITY: THE EFFECT OF pH MATERIALS REQUIRED 4A test tubes test tube rack wax pencil small mule buffer solutions of pH 10, 7, 6, 3 potato extract catecholEnayme Activity: The Bfect of pH 67 zem Posato o os g 10 « is 0 % | nea tea - tL Sue ner is fot ana. ® rious Procedue for determining which solution isan inkbtor of the enzyme catechol oxidase,Miaceach ube © Catechol Catechol Catechol Catebol added added added sdded @ tc cc re Mix eoch tube at Smite intervals fo 20 minke co FIGURE 6-5 Procedure for determining the effect of pH on enzyme activityPROCEDURE ‘The acidity, or alkalinity of a substance can be ex- ‘pressed asa numerical value, which is called pH. The PH scale ranges from 0 to 14. A pH value of 7 is ‘neutral —values less than 7 are more acidic, whereas values greater than 7 are more alkaline (see Fig. 6-5). 1, Use the males and wax pencil to mark 1 cm, 2 em, and 3.em from the boitom of 4 clean test tubes Mark the top end of the test tubes 10,7, 6, and 3 2. Fill each test ibe up to the I cm mark with the approptiate buffer solution of the same pH value a5 that written on the top of the tube 3. Fil each test tube up tothe 2 em mark with potato extract. Mix the contents well. 4, Finally leach test tube up to the 3 em mark with catechol. Mix wel. $5. Watch for color changes inthe four test tubes dur- ing the next 20 minutes. Record color changes ina table at 5-minute intervals, Use the plus and minus scale presented in Lab 6.4, Mix the solutions at Semninute intervals while ning POSTLAB QUESTIONS 1, Based on your results, what was the optinwum pH for catechol oxidase activity? 2. What effect does s high orlow pH have on enzyme activity? Why? 3, Based on your observations, why do cytoplasm and many body fluids have a pH measure near 7? Eneyme Activity The Elect of pH 69 FOR FURTHER READING ‘Alberts B, el 1989, Mateo Msg of the Cal New Yerk: Gerland Publishing. ‘Atkin; PW. 1989. 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