Enzyme Kinetics Laboratory Experiment in General Biology: BIO 111 at Morehouse College | Lab Reports Biology

General Biology Laboratory BIO 111 Morehouse College

Laboratory Study 2

Enzyme Kinetics

Objectives

1. Be able to describe the relationships between enzymes, substrates, and products

2. Evaluate the rate of an enzymatic reaction

3. Evaluate the role of substrate and enzyme concentrations on reaction rates

4. Evaluate the role of temperature and inhibitors on an enzymatic reaction

Introduction

At any given moment, a myriad of chemical reactions are occurring in a living

cell. These reactions proceed in many sets of orderly sequences and transform various

cellular compounds stepwise into a variety of products. Each reaction is catalyzed by a

specific enzyme. The word “enzyme” was coined by Kühn in 1878 from Greek words

meaning “in yeast”, reflecting the popularity of yeast research in the late 19th century.

Most enzymes can be identified by the suffix “-ase” and a prefix that usually indicates the

type of reaction the enzyme catalyzes.

Many chemical reactions do not occur spontaneously at room temperature and

one atmosphere of pressure (the pressure at sea level). However, many reactions will

proceed if temperature and/or pressure increases (energy is added) and/or if a catalyst is

added. This change occurs because an energy barrier (energy of activation) must be

overcome for any chemical reaction to proceed. A catalyst acts to lower the activation

energy by forming a complex with the reactant(s). Enzymes are catalysts of biological

chemical reactions and they lower the energy of activation in a manner similar to

catalysts of inorganic and organic (but non-living) chemical reactions. Enzymes are

generally much more efficient than chemical catalysts and are capable to acting within a

limited range of temperature and at one atmosphere of pressure.

Most known enzymes consist of proteins of one or more chains of amino acids

(there are some catalytic RNA molecules called ribozymes). Protein enzymes may range

from 10,000 daltons (1 dalton is the mass of an atom of hydrogen) to as great a several

million daltons. The shape of a given protein, its specific conformation, is a consequence

of interactions between chemical groups within each amino acid chain or between chains

that are characteristic for each functional protein molecule. In the case of enzymes, the

folding of the amino acid chain brings certain amino acids into proximity forming the

active site of the enzyme, the location where the reactant(s) bind to the enzyme and

catalysis occurs. Enzymes are generally much more specific in their binding to reactants

than are chemical catalysts. This specificity is determined primarily by the conformation

of the active site. The active site may contain sulfhydryl groups (-SH), a metal (for

example Fe, Cu, Zn), and organic compound (flavin, pyridoxal, lipoic acid), or an

organometallic compound (heme) that also participate in catalysis.

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Objectives^ Enzyme Kinetics^ Laboratory Study 2

1. 1. 1. Be able to describe the relationships between enzymes, substrates, and productsEvaluate the rate of an enzymatic reactionEvaluate the role of substrate and enzyme concentrations on reaction ratesEvaluate the role of temperature and inhibitors on an enzymatic reaction Introduction cell. These reactions proceed in many sets of orderly sequences and transform various At any given moment, a myriad of chemical reactions are occurring in a living cellular specific enzyme. The word “enzyme” was coined by Kühn in 1878 from Greek words meaning “in yeast”, reflecting the popularity of yeast research in the late 19 Most enzymes can be identified by the suffix “ compounds stepwise into a variety of products. Each reaction is catalyzed by a-ase” and a prefix that usually indicates theth (^) century. type of reaction the enzyme catalyzes. one atmosphere of pressure (the pressure at sea leve Many chemical reactions do not occur spontaneously at room temperature andl). However, many reactions will proceed if temperature and/or pressure increases (energy is added) and/or if a catalyst is added. This change occurs because an energy barrier (energy of activation) must be overcome for any chemical reaction to proceed. energy by forming a complex with the reactant(s). Enzymes are catalysts of biological A catalyst acts to lower the activation chemical reactions and they lower the energy of activation in a manner similar to catalysts of inorganic and organic (but non generally much more efficient than chemical catalysts and are capable to acting within a limited range of temperature and at one atmosphere of pressure.-living) chemical reactions. Enzymes are (there are some catalytic RNA molecules called ribozymes). Protein enzymes may range from 10,000 daltons (1 dalton is the mass of an atom of hydrogen) to as great a several million daltons. The shape of a given protein, its specific conform^ Most known enzymes consist of proteins of one or more chains oation, is a consequencef amino acids of interactions between chemical groups within each amino acid chain or between chains that are characteristic for each functional protein molecule. In the case of enzymes, the folding of the amino acid chain brings certain amino a active site of the enzyme, the location where the reactant(s) bind to the enzyme andcids into proximity forming the catalysis occurs. Enzymes are generally much more specific in their binding to reactants than are chemical catalysts. This specificity is of the active site. The active site may contain sulfhydryl groups ( example Fe, Cu, Zn), and organic compound (flavin, pyridoxal, lipoic acid), or an determined primarily by the conformation-SH), a metal (for organometallic compound (heme) that also participate in catalysis.

substrate (S) is converted to a single product (P) in a reaction catalyzed by an enzyme (E), the reaction occurs by the formation of an enzyme active site. The product is freed from the active site as the catalysis is completed. This^ The reactant(s) of an enzymatic reaction is called the substrate. When a given-substrate complex (ES) at the reaction sequence is shown below (Eq. 1) and is based on the formulation of L. Michaelis and M.L. Menten (1913). changed in the reaction process. As indicated in the equation above (Eq. 1), the same enzyme can react with many substrate molecules successively as soon as it releases product. A typical enzyme reaction in the laboratory may have substrate concentrations that exceed that of the enzyme by a factor of thousands. Although the enzyme forms a complex with the substrate, the enzyme is not transformed into another compou^ Catalysts are defined as substances that act at low concentration without being!^ S^ +^ E^ "^ ES^ "^ E^ +^ P nd by the the^ Eq. 1 reaction. cell-free preparation. The evaluation of an enzyme isolated from a cell is an Assessing enzyme activity generally requires that we isolate a given enzyme in a in vitro preparation (meaning literally “in glass”, as opposed to activity may be quantified by measuring the rate of substrate disappearance or product formation. In this study, you will measure the activity of the enzyme phenoloxidase. This enzyme is also known as polyphenoloxidase (in the plan in vivo t literature) and tyrosinase, “in life”). Enzyme (in the animal literature). Plants contain large amounts of phenolic compounds in the cell vacuole and phenoloxidase in the cytoplasm. When cells are damaged by herbivores, these mildly toxic phenolics are converted to quinon animals and microbes. The products of phenoloxidase reactions also participate ines that are particularly toxic to forming a protective barrier at the site of bruises and cuts in plant tissues. In animals, this same enzyme is responsible for the formati variety of animals including humans) and the tanning response of human skin to UV exposure. on of melanin (a dermal pigment in a wide-light substrate, catechol, is oxidized b quinone, is chemically reactive and undergoes secondary oxidations and condensations, forming black^ The type of reaction catalyzed by this enzyme is shown below (Figure 1). The-brown colored products. The products of the secondary reactions thaty molecule oxygen. The product of this reaction, ortho- form colored products are rapi spectrophotometer. d so their appearance can be easily measured with a

On your laboratory bench Spectrophotometer Rack containing 4 glass cuvettes Rack to hold test tubes of buffer and catechol Small flask or beaker to hold deionized water Styrofoam cup for ice bath Pipette helper On supply bench Test tubes for reagents Disposible graduated plastic pipettes (1 ml and 5 ml) Pieces of parafilm Crushed ice in a cooler Enzyme preparation in Stock solution of 0.05 M phosphate buffer at pH 7.0 Stock solution of 0.01 M catechol refrigerator (prepared this morning) Stock solutions of inhibitors Boiling water bath on a hot plate and test tube forceps Frozen 1ml samples of enzyme in microfuge tube Styrofoam cups for use as hot water baths s beginning the experiments:^ Dial face metal stem thermometers^ Each group will need to obtain the following from the supply bench before 30 ml phosphate buffer in a large test tube (label your tube) 15 ml cate 5 ml pipettes (3) and 1 ml pipette (1) several pieces of parafilmchol in a test tube (label your tube) fill the cup from your bench with crushed ice 15 ml of enzyme preparation in a tube (keep on ice) card indicating the additional experiment you should conduct Warning toxin at the concentrations we are using. Wipe any and all spills immediately and inform Use care in this experiment and the others in this study. Catechol is a moderate your instructor. Wash your hands after this laboratory and b Experimental Protocol efore eating or drinking. values as a function of time. The times will be 30s, 60s, 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min, 10min.^ Before you begin, prepare a data table in which you may record absorbance

containing the following components for the trial run (Table 1): Table 1. The quantities of reaction components to be combined in cuvette for a trial run. The final^ The total volume in each reaction mixture will be 5 ml. Prepare a cuvette component, 1.0 ml of enzym Component 0.05 M K 0.01 M catechole preparation, should be added just before the reaction is to be evaluated.-phosphate buffer, pH 7.0 Quantity (ml)2.01. Place a square of parafilm over the mouth of the cuvette tube, press w invert the tube several times to thoroughly mix. Use this tube as your Blank to calibrate^ Deionized water^ 1.0 ith your thumb, and the spectrophotometer. Once the spectrophotometer is calibrated, add 1.0 ml of enzyme preparation to the tube. Quickly, use parafilm to invert absorbance enzyme was added to the cuvette, so have your partner keep time and call out the seconds on the spectrophotometer. This first reading should be 30 seconds after the the tube once, and read the are you approach 30 s. Remove the cuvette after th make your next reading exactly 30 s after the first reading (one minute after the enzyme was added). Place the cuvette in a rack on your bench between readings (not in your fist). Do not invert the cuvette between readings. Make additional readings every minutee first reading, but keep time and for the next 10 minutes, but remove the cuvette between readings (why?). What was the color of the reaction mixture at the end of 10 minutes compared to the start of the reaction? You may pour the contents of t carefully rinse the cuvette with DI water. You may rehis trial run reaction mixture down the drain and-use this cuvette for other reactions. time. Which is the dependent vari axis)? Determine the slope of the line that is formed by the first few readings to evaluate the initial reaction rate. Does the reaction rate decrease over the 10 minutes of your^ Using graph paper (or MS Excel) plot the reactionable (y-axis) and which is the independent variable (x^ absorbance^ as a function of - experiment? Both of you should be comfortable performing either role in this experiment. You and your partner should switch roles and re-run this trial run experiment. actually an enzymatic reaction Discuss this as a class and then run an experimental control. What do you think caused the reaction rate to slow over time? What experiments could be conducted to test your^ How do you know that the color change observed in the reaction mixture was? What would be a suitable control for this experiment? hypotheses? Effect of Enzyme Concentration enzyme were to increase or decrease? What do you predict given the enzymatic process described in Eq. 1? This experiment will permit you to test your predict^ How would the rate of an enzymatic reaction change if the concentration ofions.

Prepare a graph of the reaction rates as a function of substrate concentration (use the values you calculated to complete the table above (Table 3). How do the observed results compare to your predictions? Put your rate and substrate v calculate the respective concentrations themselves) on the chalkboard to share with theolume data (everyone can class. Effect of Temperature on Enzyme Activity including enzymatic period studying temperature effects, so we will only evaluate some extremes. The treatments that are suggested are given in the table below (Table 4). You will evaluate^ Temperature has dramatic effects on the rates of most chemical reactionsally catalyzed reactions. You could easily spend an entire laboratory the effect of and process (0°C, 25°C, and 35°C). What outcomes do you predict for each treatment given your kn – 80°C), and the effect of reaction mixture temperature at the time of the reactionowledge of temperature effects on chemical processes and on living systems? pretreatment temperatures on the activity of the enzyme preparation (+100°C Table 4. Treatments of enzyme preparations and reaction mixtures to evaluate temperature effects on enzyme activity. Every treatment has the same components and volumes as the t Table 1). Treatment Pre-treatment of enzyme Temperature of Reactionrial run experiments (see Control Zero Warm 1 ml enzyme to 25°C in a pyrex tube 5 minutes prior to useNone (keep on ice) Room Temperature 25°CKeep reaction mixture on ice, prior and d (readings at 2 min intervals)uring reaction One Hundred^ Thirty-five^ Warm 1ml enzyme to 35°C in pyrex tube 5^ minutes prior to useHeat 1 ml enzyme to 100°C in pyrex tub minutes prior to use e 5^ Keep reaction mixture at 35°C,^ prior and during reaction^ (readings at 30s intervals) Room Temperature 25°C Minus Eighty Frozen 1ml enzymes in microfuge tube must be thawed prior to use (thaw gently, do not heat the microfuge tube) Room Temperature 25°C supply table. Label each tube with a treatment listed in the above table (Table 4). Pipette 1ml of enzyme into each tube except the Minus Eighty treatment. Place each pyrex tube in your crushed ice bath until you are ready to conduct^ Prepare for this experiment by gathering five small pyrex a given experimental pre^ test tubes from the- treatment. A microfuge tube containing 1ml of enzyme preparation that was frozen at 80°C may be obtained from the laboratory freezer (as your instructor). Let the microfuge tube thaw at room temperature and then pour the content “Minus Eighty” to continue thawing. When fully liquid, place the tube in the crushed ices into the pyrex test tube labeled – bath. Keep enzymes on crushed ice until ready to treat (“Control”, “Zero”, and “Thirty five”) or after pre Prepare a total of five cuvettes, labeled with the five treatments (use label tape), in-treatment until ready to use (“One-hundred” and “Minus Eighty”). - the same manner as in the trial run experiments. Each tube should contain 2.0 ml

phosphate buffer, 1.0 ml catechol, and 1 Hundred” and “Minus Eighty” tubes in a rack on your bench, to hold a room temperature (approximately 20° tube in a 35°C water bath (see below) fo-25°C). Place the “Zero” tube in crushed ice, and the “Thirty.0 ml DI water. Place the “Control”, “Oner at least 10 minutes. You will create a 35°C-five” water bath by running tap water to the luke thermometer). Fill a styrofoam with this tap water and then cover the cup with a blue form lid containing three holes (tw you place the cuvette in the 35°C water bath, check the temperature to ensure that it iso for test tubes and one for the thermometer). Once-warm temperature of 37°-38°C (check with a between 34° wipe moisture off the cuvett return the cuvette to its treatment temperature between readings.- 36°C. When you run the “Zero” and “Thirtye before placing it in the spectrophotometer, and always-five” treatments, remember to components except the enzyme, mix, add the enzyme, mix, 2min to 10min after adding the enzyme (unless suggested otherwise, see Table 4). Run one reaction tube at a time.^ Conduct these experiments in the same manner as the trial runs, combine all read absorbance at 30s, 60s, Prepare a graph of the reacti reaction rate of each experiment (E “Control” treatment (C^ Graph your results and estimate the reaction rates from the first 3r) (Eq. 2).on rates as a function of temperature treatment. Calculate ther) as a percentage of the reaction rate observed in the-5 readings.

! How do the observed results compare to your predictions? Put your rate and temperature^ E^ Crr^ "^100 Eq. 2 treatment data (everyone can calculate the respective percentages themselves) on the chalkboard to share with the class. Effect of Enzyme Inhibitors that selectively combine with the active site or a functional component of the active site (for example a metal atom, or a sulfhydryl group), or interfere with the binding of substrate. This experiment will give you an oppo^ Much of our understanding of enzyme functionrtunity to evaluate two inhibitors,^ comes from studies of chemicals phenylthiourea (PTU) and para on phenyloxidase. The chemical characteristics of these compounds and two other inhibitors are given in below (Table 5). Note that PHB catechol (Figure 2), so it may be able to bind to the active site of phenyloxidase. PTU-hydroxybenzoate (PHBA) to determine how the might actA is structurally similar to specifically binds to copper, so inhibition by PTU would suggest a copper atom at the active site of phenyloxidase.

percentages the does the active site of phenoloxydase contain Cu? What would you predict to be the inhibitory effects of KCN or K^ Put your rate and inhibitor treatment data (everyone can calculate the respectivemselves) on the chalkboard to share with the class. Given your findings, 3 AsO 3 on this enzyme? In view of your findings with PHBA, hypothesi enzyme evaluate your hypotheses? Was PHBA competing with catechol for the active site or was it binding irre-substrate complex forms. What additional experiments would be necessary toversibly?ze how catechol is oriented at the active site of phenoloxydase when an Results Summary Assignment data that is posted by your classmates. You must address all five parts of this enzyme Each of you will prepare a results summary on this entire study using the class study. Literature Cited Michaelis, L. and M 49:333-369.. L. Menten. 1913. Die kinetik der invertinwirkung. Biochem. Z. There are many good web sites on enzyme action and the rates of enzymatic reactions. The following sites are very detailed but are worth visiting for additional information on th http://www.tiem.utk.edu/~gross/bioed/webmodules/enzymes.htme Michaelis-Menten equation. http://nsr.bioeng.washington.edu/Software/DEMO/MICMEN/ Acknowledgements Action: Catalytic Properties of Phenoloxidase, in Biology 105 Course Pack, Division of Biological Sciences, University of Michigan, Ann Arbor. This study was reprinted with revision from H. Ikuma, P. Harley and C. Yocum (1985) Enzyme Revisions made 2/2005 by L. Blumer.

Enzyme Kinetics Laboratory Experiment in General Biology: BIO 111 at Morehouse College, Lab Reports of Biology

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Download Enzyme Kinetics Laboratory Experiment in General Biology: BIO 111 at Morehouse College and more Lab Reports Biology in PDF only on Docsity!

Objectives^ Enzyme Kinetics^ Laboratory Study 2