Enzyme Reactions Skill Check Biology Worksheet

Enzyme Reactions Skill Check Biology Worksheet

Activity 1: Determining the Reaction Rate in the Presence or Absence of Cellobiase Data Table 1: Comparison of Reaction Cuvettes to Standard Cuvettes. Time (minutes) Cuvette 0 8 1 2 4 6 8 START END E1 E2 E3 E4 E5 Standard that is Most Similar S1 S1 S2 S3 S4 S5 S5 Amount of p-nitrophenol (nmol) Cuvettes after mixing enzyme sample with stop solution Calculation of Initial Rate of Product Formation (with enzyme present) Calculation of rate of Product Formation with no enzyme present Page 1 Skill Check Ex.9: Introduction to Enzyme Reactions Activity 1 Questions 1. Describe any changes you observed in the enzyme reaction and control reaction conical tubes during the time that the reaction was occurring? 2. What happened to the solution in each cuvette after you added the enzyme/substrate mixture to the stop solution that was in the cuvette? 3. Describe the amount of product produced in the enzyme-catalyzed reaction compared to the control where no enzyme was added. 4. If you took another time point at 15 minutes, do you think more product would be produced than at 8 minutes? Explain your answer. 5. Is the rate of product formation constant over time? (Hint: Is the slope of the line constant or does it change over time?) Page 2 Skill Check Ex.9: Introduction to Enzyme Reactions BE SURE TO ATTACH YOUR GRAPH FOR THIS ACTIVITY TO THE SKILL CHECK WORKSHEET WHEN YOU TURN IT IN! Activity 2: Determining Effects of Temperature on Cellobiase Activity Data Table 2: Determination of ρ-nitrophenol produced at three different temperatures based on ρ-nitrophenol standards Temperature Standard that is most similar 0C Room temp 37C S2 S3 S5 Amount of ρ-Nitrophenol Produced (nmol) BE SURE TO ATTACH YOUR GRAPH FOR THIS ACTIVITY TO THE SKILL CHECK WORKSHEET WHEN YOU TURN IT IN! At what temperature does this enzyme work best? Explain how you know this. Why do chemical reactions occur faster at higher temperatures? Is that true for all enzymes, and unlimited temperature values? Why do most enzymatic reactions slow down at extremely high temperatures? Page 3 Skill Check Ex.9: Introduction to Enzyme Reactions Activity 3: Determining Effects of pH on Cellobiase Activity Data Table 3: Determination of ρ-nitrophenol produced at three different pH’s based on pnitrophenol standards pH 5.0 6.3 8.6 Standard that is most similar S3 S2 S1 Amount of p-Ntrophenol Produced (nmol) Activity 3 Questions a. At what pH does this enzyme work best? Explain how you know. b. Why do most enzymatic reactions slow down at extremely high or low pH values? c. In what type of environment might an organism that produces this enzyme live? Explain your reasoning Activity 4: Summary ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ Page 4 Skill Check Ex.9: Introduction to Enzyme Reactions ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ Page 5 Skill Check Ex.9: Introduction to Enzyme Reactions ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ _____________________________________________________________________ Page 6 Skill Check Ex.9: Introduction to Enzyme Reactions Page 7 Skill Check Ex.9: Introduction to Enzyme Reactions Pre-Lab Exercise 9: Introduction to Enzyme Reactions Introduction Living organisms depend upon the thousands of chemical reactions that occur within them for their existence. These reactions, if left on their own, would occur very rarely, making life impossible. Catalysts are molecules that speed up the rate of chemical reactions without being destroyed or altered in the process. Enzymes are biological catalysts that speed up the rate of the biochemical reactions in living organisms. They are not permanently altered in the process, and can be used over and over. Most enzymes are globular proteins that exhibit tertiary (Figure 1), and in many cases, quaternary structure. The enzyme’s function is dependent upon the three dimensional shape. Each enzyme has a substrate, the molecule or molecules to be acted upon in the chemical reaction. You can also think of the substrate as one or more of the “reactants” in the chemical reaction. The substrate binds reversibly to the active site of the enzyme molecule. The active site of the enzyme is constructed based upon the amino acid sequence of the protein, and as such, each has a unique three-dimensional structure. In most cases only a single type of substrate, or a small group of closely related substrates, can bind to the active site of a specific enzyme. Anything that disrupts or blocks the three dimensional structure of the active site of the enzyme will affect the activity of the enzyme. When the three dimensional structure of the enzyme is altered, the enzyme is said to be denatured. Enzymes are generally given common names that relate them to the substrates that they act on, and the names usually (but not always) end in the letters –ase. For example, the enzyme that would break down sucrose into glucose and fructose is called sucrase. Figure 1: Structural Model of the enzyme Cellobiase Enzymes are globular proteins that have at least tertiary structure. The function of the enzyme depends upon the specific three-dimensional shape, one part of which (the active site) is designed to “fit” a particular molecule, the “substrate.” If the shape of the active site of the enzyme is disrupted or blocked, the enzyme will not be able to function normally. In order to understand how an enzyme works under different conditions, scientists study the enzyme’s kinetics, the rate at which an enzyme works. For examples, one molecule of the enzyme catalase can break down millions of molecules of H2O2 per second. But this is the maximum rate at which the enzyme can operate, and there are a number of different factors that can affect this rate including: q Concentration of enzyme: If the amount of substrate available to an enzyme is unlimited, then the rate of the enzyme reaction will change with different amounts of enzyme. q Concentration of substrate (in this experiment, the amount of H2O2 present): if all other conditions are constant, the rate of a reaction should increase with increasing concentrations of substrate. At very low amounts of substrate, the reaction rate will increase rapidly. Eventually, at higher substrate concentrations, the rate will level off and eventually the maximum rate for that reaction will be reached and further increases in the concentration of substrate will have no effect. q pH of the enzyme’s environment: Changes in pH alter the state of ionization of charged amino acids such as lysine, aspartic acid and glutamic acid. As the pH environment of an enzyme moves into the acidic range, the enzyme will tend to gain hydrogen ions from the solution, while in the basic range, those hydrogen ions will be lost to the solution. Changes in ionization may result in changes in the shape of the enzyme, which in turn may affect binding of the substrate to the enzyme, or may directly change the enzyme’s catalytic action. q Temperature: Hydrogen bonds are easily disrupted by increasing temperature, and changing those bonds may result in a change in the shape of the enzyme. Such shape changes may decrease the affinity of the enzyme for its substrate. This denaturation of the enzyme results in a loss of catalytic activity for many enzymes at high temperatures. q Salt concentration: every enzyme has an optimal salt concentration in which it can catalyze reactions. Salt concentrations that are too high or too low will denature the enzyme. q The presence of inhibitors: the presence of competitive and noncompetitive inhibitors can affect the enzyme’s activity. Copper sulfate is a noncompetitive inhibitor of catalase, while cyanide is a competitive inhibitor that binds to the active site in the catalase molecule. Every enzyme has an optimal range for each of these factors, and catalytic activity of the enzyme will decrease under conditions that are outside of the optimal range. In the lab exercise this week, you will examine three of these factors: concentration of enzyme, pH and temperature. Cellobiase: Part of the Beta-glucosidase family of enzymes Cellulose is the structural polysaccharide found in the cell wall of plants. It can be used as a source of sugar for organisms that produce a family of enzymes know as cellulases. Cellobiase (EC is a type of cellulase that catalyzes the breakdown of cellulose to glucose and falls into a family of enzymes known as beta (b)-glucosidases. These enzymes break down substrates that are connected together by a 1,4 b-glucoside linkage. Cellobiase will break down cellobiose, a disaccharide made up of two glucose molecules connected together by a 1,4 b-glucoside linkage. Humans and other animals do not produce cellulases, but many plant-eating animals are hosts to other organisms that do possess these enzymes. For example, termites carry the protozoan Trichonympha living inside their gut (Figure 2). Trichonympha has a bacterium living inside it that produces cellulase enzymes that break down cellulose, the main component of wood. Ruminants such as cows harbor anaerobic microorganisms that digest the plants they eat. Many types of fungal decomposers derive much of their food from the cellulose in the cell walls of plants they digest. The filamentous fungus Aspergillus niger produces cellulases that it secretes from its hyphae to digest cellulse in its surroundings to use as a food source. C Figure 2: Termite (a) protist Trichonympha (b) that lives in its gut and cellulose digesting bacteria (c) that live in Trichonympha Cellulosic Ethanol: A Practical Application for Cellulases The biofuel industry uses cellulases to convert the cellulose in plant cell walls to sugars, such as glucose. The sugar can then be converted to ethanol by microbial fermentation. The ethanol can in turn be used alone in certain engines, or in combination with gasoline to power car or other engines. A plant’s biomass is mostly cell wall material that is made up primarily of cellulose, a long chain of glucose molecules. Each cellulose molecule is attracted to other cellulose molecules by hydrogen bonds, resulting in the formation of microfibrils made up of 60-80 individual strands of cellulose. Plant cells can be alive or dead at maturity. The ones that are dead strengthen the plant and/or function to conduct water through the plant. These plant cells develop a second type of cell wall called a secondary cell wall before they die. Secondary cell walls are more rigid than primary cell walls and contain additional molecules other than cellulose that contribute to their rigidity. Hemicellulose and lignin are found in high quantities in the secondary cell walls of woody or fibrous plant tissue. For cellulosic ethanol production, lignins must be removed because they inhibit enzymatic activity of cellulases. Hemicelluloses must be cleaved from the cellulose to allow enzymatic breakdown of the cellulose. The production of ethanol from plant material is a very complex procedure requiring multiple steps. Plant material is first processed mechanically, as well as with acids and enzymes and heat to remove lignin. Once the lignin is removed, the cellulose can be more readily broken down. It is broken down into glucose in three steps by three different types of enzymes: Endocellulases: these enzymes break down the internal bonds of the long chains of glucose molecules that form cellulose. + H2O + Exocellulases: these enzymes break the covalent linkages between the glucose units of cellulose that are on the end of the cellulose molecules, releasing cellobiose. + H2O + Cellobiases (b-glucosidases): these enzymes break down the cellobiose left behind as a result of the work of the first two enzymes (this is the reaction we will measure in this lab exercise!). + H2O + Cellobiase Cellobiose Glucose Glucose Once this series of pre-treatment and enzymatic hydrolysis steps have been completed, the remaining mixture can be fermented by other microorganisms to produce ethanol. Assaying Cellobiase Activity In order to study an enzyme there must be a way to measure its activity. There are a number of ways to achieve this, generally by measuring the production of a product of the reaction or the disappearance of a reactant. Although cellobiose is the natural substrate of cellobiase, there is no simple method to quantitatively detect production of the product (glucose) or the disappearance of cellobiose. To make this reaction easier to follow, a simple colorimetric assay (an assay that produces a color that can be seen or measured) can be done using an artificial substrate. In this case the artificial substrate рnitrophenyl glucopyranoside can be used to detect enzymatic activity of cellobiase. This artificial substrate can also bind to the enzyme cellobiase and be broken down in a manner similar to the natural substrate cellobiose, The substrate р-nitrophenyl glucopyranoside is composed of a beta glucose covalently linked to a molecule of ρ-nitrophenol. Then the bond connecting these two molecules is cleaved with the help of cellobiase, the р-nitrophenol is released. cellobiase H2O + Artificial SUBSTRATE p-nitrophenyl glucopyranoside + Glucose PRODUCTS + p-Nitrophenol To stop the activity of the enzyme (by denaturing it) and to create a colored product, the reaction mixture is added to a strongly basic solution. When the р-nitrophenol is placed in a basic solution, the hydroxyl group on the nitrophenol loses an H+ to the OH- of the base, which changes the bonding within the phenolic ring so that the molecule will absorb violet light and reflect yellow light. This makes the solution yellow, which can be detected visually by comparing the deepness of the yellow color to a set of standards of known concentration of р-nitrophenol or by using a spectrophotometer to produce more accurate, quantitative results. The amount of yellow color is proportional to the amount of р-nitrophenol present. The deeper the color, the greater the amount of product produced by the enzyme. In this lab exercise you will be comparing your experimental samples to a set of known standards of рnitrophenol in order to estimate the amount of р-nitrophenol produced during the enzyme reaction. Each of the prepared standards contains a known amount of р-nitrophenol and will have a yellow color. The more intense the yellow color, the greater the amount of р-nitrophenol in the tube. One way to estimate how much product has been produced is to compare the yellowness of the enzyme reaction samples to a set of standards which contain a known amount of р-nitrophenol product. This in turn is proportional to the amount of cellobiase enzyme activity. In order to understand the factors that influence an enzyme’s ability to break down its substrate, the rate of reaction, (how much product is formed in a set amount of time), is determined. For studying cellobiase activity you will measure the rate of reaction by adding enzyme to the artificial substrate p-nitrophenyl glucopyranoside. The enzyme and substrate are initially dissolved in buffer that is at an ideal pH (pH 5.0) for the reaction to occur. At set times, a sample of the enzyme reaction will be removed and added to a high pH (basic) stop solution which will develop the color of the product p-nitrophenol, as well as stop the reaction by increasing the pH to above the range where the enzyme can work. By calculating how much p-nitrophenol is produced over time, the rate of reaction can be calculated. By looking at small increments of time, you will be able to determine whether the rate of the enzyme is constant or whether it slows down as the amount of substrate decreases. You will also be able to detect any effects pH or temperature might have on the reaction, thus characterizing the overall properties of the enzyme. Exercise 9 Enzyme Kinetics Introduction • Enzymes are proteins which expedite chemical reactions • Enzymes are catalysts • Catalysts speed up chemical reactions and are unchanged after the reaction allowing a single enzyme to carry out many chemical reactions • Enzymes carry out many types of chemical reactions: • Examples: DNA synthesis, RNA synthesis, protein modification, amino acid synthesis, energy utilization reactions, carbohydrate synthesis, protein degradation and many more. • In this lab we study an enzyme which breaks down a disaccharide (cellobiose) into two monosaccharides (glucose). • The enzyme is called cellobiase Studying the enzyme kinetics of cellobiase • Lets say you are working for a biotech company developing ways to develop glucose manufacturing techniques. Glucose could then be sold to other companies for other applications like alcohol production for clean energy vehicles. Some plants, like corn, could actually be a cheap source of glucose. • Cellulose is a polymer of glucose. The success of yours and other companies may depend on your ability to generate inexpensive glucose from this cellulose • The enzyme cellobiase is an enzyme you need to understand as it is involved in producing glucose. Enzyme Kinetics • We need to know the optimum conditions for cellobiase activity. • Which conditions provide maximum rate (speed) of reaction • Factors affecting enzyme kinetics (speed) • • • • • • Temperature pH Salt concentration Enzyme concentration Presence of inhibitors Substrate (reactant) concentration • In this lab we will study the affects of temperature and pH on cellobiase activity Measuring Cellobiase Kinetics • We need a way to measure the cellobiase reaction • Generally speaking: Enzyme + Substrate → Product + Enzyme • This is a chemical reaction except we call the reactant a substrate in an enzymatic reaction. Al…


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