Student Guide Name Carolina™ Evolving Enzymes for AP Biology Background fora chemical reaction to occur reactants must code with enough enery to break her chemical bonds, Slowing new bonds tobe formed inthe product, Most chemical reactions in ving organisms would ge too sow to nstain fe i were nt for specialized protein called enzymes. Enzymes ac as catalysts {anced up chemical reaction by lowering the activation energy required forthe reaction Figure). ‘Ketration energy the minimum amount of energy equired to tara chemical renion. ‘gure 1. raph doping the activation energy for raaton with and without a catalyst ‘An enzyme often binds to one ofthe substrates ina way that changes the substrate’ shape, making it more fikely to react with another substance. An enzyme might instead bind moltiple substrtes, bringing them closer together and thereby making them more likely to react. The action of an enzyme is specific toa reaction ota set of reactions: each enzyme binds exclusively tothe substrate or substaes of the reactions) ‘eeatalyzes. This specificity occurs because the shape of the enzyme fit or interlocks withthe shape of one ‘or both ofthe subtrates, forming what i called the enzyme-substrate complex. Like other catalysts, an ‘enzyme remains unchanged by the reaction. When the reaction fs completed end the enzyme isno longer bound to the substrate), the enzyme returns tots original form. The analogy of a lock ‘and key was historically used to describe the fenzyme-substrate relationship. The enzyme has a binding pocket, called an active site, ee CO_ dA pomreni replaced by the “induce fit” model, which ‘count forthe potential shape change of “ce the enzyme wien it binds to its substrate. Figure 2. tlustration of the enzyme-substate complex ce cnt ey mek congue = S-1volung Enzymes Kit or AP Bioloy Student Guide Various biotic and ablotk factors influence the activity ofan enzyme. An enzyme operates best within 2 narrow range of tempercture, concentration, salinity, and pH peculiar to the biological system in which it functions For example, several enzymes break down food inthe human digestive system. Different, enzymes operate in different pars of the digestive tract, Some parts of the digestive tract are acidic (oH less than 7), whereas others ar alkaline (pH greater than 7). The stomach contains adc gastric juices ‘hat aid with digestion. Enaymes that are active in the stomach function best n that acidic environment. Food exits the stomach tothe small intestine. The alkaline smal intestine neutralizes the acified food coming from the stomach, Enzymes active inthe small intestine function best in an alkaline environment, \When pit values are outske the optimum range, the enzyme may denature, or its acive site may change conformation. Enzymes active inthe stomach are generally inactive inthe small ntesine, and vice versa. ‘There may be other molecules in an enzyme's envionment that help or hinder the ality and efficiency ofthe enzyme by binding tothe substrate, Coenzymes and cofactors are activators tat increase reaction levels. Coenzymes include small inorganic compounds and ions, while cofactors include organic molecules such a vitamins. Inhibitor molecules may block the ative site from the substrate permanently or only temporarily. Many inhibitors are produced and used by organisms to regulate thelr normal reactions. introduced drugs or toxins may aio inhibit enzyme function. Competitive inhibitors are molecules that have a charge and conformation allowing them to block the active site of the enzyme rom the substrate. Competitive inhibitors afect the initial rate of reaction but rot the maximum rate. A noncompetitive inhibitor most often binds to an enzyme away from the active site; however, this binding results in a conformational change that prevents the binding ofthe substrate to the ative site. Noncompetitive inhibitors reduce the maximum rate of reaction. Bioinformatics ioinformatics isthe field that identifies biological information in DNA and amina acid sequences using ‘computer-based tools. Te identification of sequences allows users to compare genes, proteins, and other functional elements of DNA among organisms and species. Because of the large number of tools and DNA sequences available on the Internet, theoretical and analytical experiments done on the computer often complement experiments dane in the laboratory Enzyme Evolution ‘The enzyme catalase is ubicutous in aerobic organisms. As organisms evolved to use oxygen for cellular respiration, the ability to quickly break down hydrogen peroxide, a pokonous by-product of cellular respiration, was advantageous. The catalyzed decomposition of hydrogen peroxide produces harmless water and oxygen gas: 2H,0, 285 2H.0+0, Species that have similar DNA sequences evolved from a common ancestor. Organisms that are more distantly related have, overtime, accumulated more differences in thelr DNA sequences via mutations. “The variation between the amino acid sequences ofa protein (uch as catalase) from two different ‘organisms can indicate how closely those organisms are related. ‘Variation i caused by mutaions. Harmful mutations that hinder an organisms fitness, its ability to survive and reproduce, ar ls likely to be passed on to the next generation, Mutations that generate advantages ‘may be passed along through natural selection, the driving force of evolution, acannon Cangui® = S-2Evolving Enzymes Kit for AP Biology Student Guide Mutations may be caused by mistakes during DNA replication or result from environmental influences such cUV radiation or chemical exposure, Many types of mutations exist, including point mutation, insertion, ‘Jeleton, ond gene duplication. Point mutation isthe replacement of one nucleotide base with another: castor occur when nucleotides ae added into a DNA sequence, Deletion occurs when nucleotides are arated from a DNA sequence, Gene duplication, a specific kind of insertion, occurs when an entire section SFONA in the genome that codes for one protein i mistakenly copied twice. fall the regulatory sequences het control transcription and transation of the protein are present inthe duplication, both regions aa for tha same protein. With a second copy ofthe gene available, one sequence may change without Segatively affecting the organisms ftnes. The duplicate gene sometimes mutates and serves 2 pew funetion without necessarily harming the organism. daca ot pin wreagrg wane widest a tae hee fin st sas man in eran, wih ot ce ee a tertre ONA moe oh a 2A na ines cement ro a a a cat ontangse a a ae yc nnayrn > ce a eta anaemic Organisms share genes from common ancestry. The variation between the amino acid sequences of two matching proteins (proteins coded for by the ame gene) from two different aoe ‘organisms can indicate how closely those organisms are related. ‘The relationship among genesis most easly visualized using a phylogenetic tree diagram, which may be based on ONA, RNA, er for amino acid sequence. Figure 2, Gane duplication ona chromosome Overview ‘You wil investigate the protein catalase, 8 common enzyme found in most arabic organisms. Catalase ‘decomposes hydrogen peroride, a toxic by-product of cellular metabolism, before ithas a chance to damage the cell The reaction produces harmless water and onygen gas. Se the equation below. 21,0, —> 240 +0, ‘Your group will perform an experiment t help determine the optimal conditions forthe catalase-aided decomposition of hydrogen peroxide. Each group will alter one of the following vaiables—enzyme Concentration, temperature, pl, or substrate concentration—and determine the effecton the reaction ‘ste. Groups will then share thei results, The rate of reaction willbe quantified indirectly by measuring the ‘nygen product ofthe reaction. Fite paper disks that have been treated with catalase willbe place in 2 salaton of hydrogen peroxide. As the reaction proceeds, oxygen gas forms onthe fter paper isk, causing the dst to float. The amount of time unt the disk floats is ued as a measure of reaction rate for the ‘enzymatic reaction.von Enaymes Kit fOrAPBislooy Student Guide Pre-laboratory Questions 1. White a balanced equation forthe dacampasition of hydragen pero 2. Name the following participants in catalase decomposition of hydrogen peroxide, Substeat b. Enzyme: Intermediate complex that formed (nat observed): Pender 2. List three factors that might affect the activity ofthe catalate end explain each ofthe effects Guided Activity Materials _2well microplate access to tap water Small medicine cups access to 1.5% hydrogen peroxide ‘large medicine cup (for hydrogen peroxide) access to 400-Uimk catalase 6 transfer pipets access to 40-Uinl. catalase forceps access to 4-Uiml catalase ‘medicine cup with 30 filter paper disks access to 0.4-UimL catalase timing device paper towels permanent marking pen cea ga em Orman nh CARgUN =| _S-4Folving Enzymes Kit or AP Biology Student Guide Procedure 1. Answer the Pre-laboratory Questions 2, Read the entre procedure forthe Guided Activity, 3. Label five small medicine cups tap water, 400-Ulmk catalase, 40-UIm. catalase, 4-UlmL catalase, and O.4-Uimt catalase, 4, Label the large medicine cup 1.5% hydrogen peroxide 5. Bring the labeled medicine cups tothe central materials station and collec the following {60 mL 1.5% hydrogen peroxide 10 ml 40-Uim catalase 30 mL tap water 10 mL 4-Uimt catalase 10 mL 400-Uim catalase 10 mL 0.4-Uhmk catalase 6. Set up the 24-well microplate a. Notice the coordinate letters and numbers marked on the microplate. Use the letters and numbers tokeep track of what solution is in each well. lee = S4un\isnno)(sann,)(rseno,)(isxne)( mo oN a Na BS 9 eX Xo (sm) NY ( ’ (SuES ) (ten, )(t¥ me) (s94m0, (rva)l no / NIN Daa Y soume \ / [Suit )(rsH no, |(199 0,299 Ho, (t¥na)( we 7 "Figure 4. Organzation of solutions inthe mlcroplte wel Use the transfer pipet to fill wells A6, 85, C6, and DG with tap water from the small medicine cup. Use separate transfer pipets to fill wells from the small medicine cups as follows 1 with 0.4-Uiml catalase (Ct with 40-Uiml catalase 81 with 4-Uint. catalase I with 400-Uimt catalase ‘The volume in each wellis approximately 3 mt, 4. With a fresh pipet, il the rest ofthe wells with 1.5% hydragen peroxide from the large medicine cup 7. Practice using your timing device. You may have to time events that occur in less than 1 second. 4 ais erg ey Come CAROLINA = S-5Evohing Enzymes Kit for AP Biology Student Gui 8, Test your technique and check for contamination with a water dk Use the forceps to pick up one ter paper disk. Dip the iter paper disk into the AS water well and old it therefor 1 second. ‘Touch the fiter paper disk tothe edge of the well o remove any excess drips of water 4d. Prepare the timing device. Transfer the disk to the AS well of hydrogen peroxide and release the disk a the bottom of the well 1. After the disks released, start the timer. 4. Observe the disk for up to 90 seconds. f the dik floats, Fecord the time. fit does not float within 90 seconds, record the time as >90 seconds and move on tothe next step. 9, Test the reactivity of the 0.4.UimL catalase in 1.5% hydrogen peroxide. 8, With forceps, di @ new fier paper disk into well AI of 0.4-UimL cat for cond, solution and hold it there b. Touch he filter paper disk to the edge ofthe well to remave any exces drips of catalase solution ‘& Prepare the timing device. Transfer the disk to the A2 hydrogen peroxide and release the dik atthe botton of the wel, 1. After the disk is released, stat the timer, «Observe the dik and stop the timer when the disk reaches the surface of te liquid If the disk does ot rise within 80 seconds, move on to the next stp. {Recor the surfacing time or "290 s" a5 Til 1 “Catalase clk time to surface (0) in Table 1. Leave the catalase disk inthe walls that you know the well has been used, Test the 0.4-UimnL catalase twice more, sing the hydrogen peroxide in wells A3 and AA, Record ‘these times as Trial 2 and rial 3. 10, Repeat thi pattern with the wells in the second row ofthe test plate, to test reactivity of the 4UlmL catalase, 1, Using» new dis, frst repeat the water disk test atin step 8, this time with the watar from well 96 and the hydrogen peroxide from BS. . With forceps, dip a new filter paper dik into wel BY of 4-U/ml catalase solution and hold it there for. 1 second, ‘© Touch the filter paper disk to the edge ofthe well to remove any exces dips of catalase solution. {d. Prepare the timing device Transfer the disk to the 82 hydrogen peroxide and release the disk atthe bottom of the wel 12. Afterthe dick released, star the timer 4 Observe the dik and stop the timer when the disk reaches the surface of the liquid I the disk does ot rise within 90 seconds, move on tothe next step. 1, oF “> 90s" as Tal 1 "Catalase dick time to surface ()" In Table 1 Record the surfacing Leave the catalase dis in the walla indicate thatthe well hasbeen used, |. Test the 4-Uim catalase twice more using the hydrogen peroxide in wells B3 and B4. Record these ‘times 2s Tal 2 and Trial 3 ‘214 cor Be oy Cmeantrts caRQUMG =—_S-6” 2. 16. 1. Evoing Enzymes Kt for AP Blotogy student Guide Repeat this pattern with the wells inthe third row ofthe test plate, to test reactivity ofthe 40-UmL catalase. 1a. Using a new disk, frst repeat the water disk test a in step 8 this time with the water from well C6 and the hydrogen peroxide from C5. '. With forceps, dip new fiter paper disk into well C1 of 40-Uim catalase solution and hold it there for 1 second, © Touch the fiter paper disk tothe edge of the well to remove any excess dips of catalase solution, 4, Prepare the timing device. Transfer the dsk to the C2 hydrogen peroxide and release the dik atthe bottom ofthe wall (After the disk is release, start the timer 4 Observe the disk and stop the timer when the dick reaches the surface ofthe liquid. Wf the disk does not rie within 90 seconds, mave on to the next step. 19 Record the surfacing time, oF “2905” as Tal 1 “Catalase disk time to surface ("in Table 1 Leave the catalace disk in the well to indicate thatthe well has been used |. Test the 40-Urmt catalase twice more, using the hydrogen peroxide a wells C3 and C4. Record these times as Tal 2 and Trial 3 ‘Repeat this pattern withthe wells in the fourth row of the test pate, to test reactivity ofthe 400-U/mL catalase, 2 Using a new disk, first repeat the water dsk est, and the hydrogen peroxide from DS. '. With forceps, dip 2 new filter paper disk into the well D1 of 400-Uim catalase solution and hold it there for 1 secand, In stop 8 this ime withthe water from well DS Touch the fiter paper disk to the edge of the well to remove any excess crps of catalase solution. 4. Prepare the timing device. Transfer the disk to the D2 hydrogen peroxide and releae the disk atthe ‘bottom ofthe wel ‘efter the disk is released, start the timer. 4. Observe the disk and stop the timer when the disk reaches the surface of the liquid Ifthe disk does ‘not rise within 90 seconds, move on to the next sep. Record the surfacing time, oF "590" as Trial 1 "Catalase disk time to surface ("in Table 1 |. Leave the catalase dik in the well o indicate thatthe well has been used. |. Test the 400-Uimi catalase twice more using the hydrogen peroxide in wells D3 and DA. Record these times as Tal 2 and Til 3. ‘Average each set of trials and record these values in Table 1. Share your data, collect data from the other groups, and graph the data asa class, Data from groups ho conducted the same experiment may be presented on the same graph, Discuss the experimental results with your class according to your teacher instructions. cette anreent CARQUING = 5-7Evohing Eraymes Kit for AP Biology Student Guide Data Tables Table 1, Raw Data for Reaction Rate vs. Enzyme Concentration Catalase Ditation | Temperature CQ) | Hydrogen Peroxide | Wial | Catalase disk time to surface @) 4 Um 35 1.50% Taiz mi 2 1 vim * som ais Average Taal Taal 40.UNm. 23 150% ‘2, On the bass of the enzye actity graph shown, determine if candle’ inhibition of cytochrome odes is competitive oF nencampettne. expla, , Draw an exon curve ete ra wate you would expect i ler type of ihition (ompettve oF ‘pancompettive) oe caRgune —$-13 caida po cag