Enzyme Activity Instructor Guide-7414

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Montgomery College *

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Chemistry

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Apr 3, 2024

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Enzyme Activity Instructor Guide Learning Objectives Upon completion of this lab activity, the student will be able to: ● Describe the components of an enzymatic reaction. ● Communicate the function of an enzyme’s active site and its relationship to the substrate. ● Describe the relationship between an enzyme’s structure and its function. ● Explain the relationship between maltose produced and enzymatic (amylase) activity. ● Analyze the effect of environmental factors on enzymatic activity. ● Define absorbance and transmittance and utilize data obtained with a spectrophotometer. ● Describe the relationship between absorbance and concentration. ● Construct a standard curve using known amounts of maltose. ● Utilize a standard curve to determine enzyme activity of the experimental amylase. Background Adapted from https:// www.oercommons.org/courseware/lesson/56959 How Enzymes Function A substance that helps a chemical reaction to occur is a catalyst , and the special molecules that catalyze biochemical reactions are called enzymes . Almost all enzymes are proteins, made up of chains of amino acids, and they perform the critical task of lowering the activation energies of chemical reactions inside the cell. Enzymes do this by binding to the reactant molecules, and holding them in such a way as to make the chemical bond-breaking and bond-forming processes take place more readily. It is important to remember that enzymes don’t change the ∆G of a reaction. In other words, they don’t change whether a reaction is exergonic (spontaneous) or endergonic . This is because they don’t change the free energy of the reactants or products. They only reduce the activation energy required to reach the transition state (Figure 1).

Figure 1: Enzymes lower the activation energy of the reaction but do not change the free energy of the reaction Link to a video Enzymes and Activation Energy Enzyme Active Site and Substrate Specificity The chemical reactants to which an enzyme binds are the enzyme’s substrates . There may be one or more substrates, depending on the particular chemical reaction. In some reactions, a single-reactant substrate is broken down into multiple products. In others, two substrates may come together to create one larger product molecule. Two reactants might also enter a reaction, both become modified, and leave the reaction as two products. The location within the enzyme where the substrate binds is called the enzyme’s active site . The active site is where the “action” happens, so to speak. Since enzymes are proteins, there is a unique combination of amino acid residues (also called side chains, or R groups) within the active site. Each residue is characterized by different properties. Residues can be large or small, weakly acidic or basic, hydrophilic or hydrophobic, positively or negatively charged, or neutral. The unique combination of amino acid residues, their positions, sequences, structures, and properties, creates a very specific chemical environment within the active site. This specific environment is suited to bind, albeit briefly, to a specific chemical substrate (or substrates). Due to this jigsaw puzzle-like match between an enzyme and its substrates (which adapts to find the best fit between the transition state and the active site), enzymes are

known for their specificity. The “best fit” results from the shape and the amino acid functional group’s attraction to the substrate. There is a specifically matched enzyme for each substrate and, thus, for each chemical reaction; however, there is flexibility as well. Figure 2: Induced-fit model of enzyme function Created by TimVickers, vectorized by Fvasconcellos/Public domain According to the induced-fit model , both enzyme and substrate undergo dynamic conformational changes upon binding (Figure 2). The enzyme contorts the substrate into its transition state, thereby increasing the rate of the reaction. The molecule(s) produced at the conclusion of the reaction is called a product(s). Link to a video Enzymes the Induced Fit Model Factors that affect the rate of enzyme activity: Adapted from Amylase Activity Lab , Montgomery College, Rockville, MD ( doi:10.25334/VKGJ-VT46 ). 1. Temperature affects enzyme activity in two ways. As the temperature rises, molecular motion (kinetic energy) increases and the rate of random collision between enzyme and substrate molecules increases, forming more products. After a certain point, increasing the temperature strains the non-covalent bonds, altering the shape of the active site, and the overall shape of the enzyme, the enzyme is then denatured . This decreases the rate of product formation. The temperature at which enzyme activity is the highest is called the optimum temperature . At high temperature, an enzyme will most likely unfold and denature. 2. Changes in pH (H+ concentration) and salt concentration primarily affect the stability of secondary and tertiary structures maintained by hydrogen bonds and disrupt salt

bridges held by ionic bonds. As a result, enzymes denature at extreme pH and high salt concentrations. In addition, substrates and/or enzyme active site groups may ionize, which further affects enzyme substrate binding. The pH at which enzyme activity is the highest is called the optimum pH . 3. Substrate and enzyme concentration also affect the rate of enzyme reaction. Increasing the concentration of substrate and/or enzyme increases the rate of reaction up to a certain point. As the reaction continues and the substrate molecules are used up, the rate of reaction will decrease regardless of any changes in enzyme concentration. By controlling enzyme and substrate concentration, organisms can regulate their metabolism. Link to an interactive on Effect of pH on Enzyme Function Link to an interactive on Effect of Temperature on Enzyme Function Amylase Enzyme Activity: Amylase The enzyme being studied in this experiment is amylase , an enzyme which cleaves complex sugars (polysaccharides) into simple sugars (disaccharides). Amylase catalyzes the hydrolysis (splitting) of α-1,4 glycosidic linkages in polysaccharides (like starch) which breaks the large molecules into smaller molecules of sugar like maltose. Maltose is a reducing sugar that consists of two molecules of glucose bonded together. In this reaction, starch is the substrate and the product is maltose (Figure 3). Figure 3: Hydrolysis of starch into maltose by amylase From https://commons.wikimedia.org/wiki/Category:Maltose#/media/File:Amylase_reaction.png

Alpha-amylase is produced by a wide variety of organisms from all three domains of life: Bacteria, Archaea, and Eukarya. It serves to breakdown starch which is the most widely available polysaccharide on the planet. When digested, starch produces simple sugars that are readily available energy for organisms. In humans, alpha-amylase is a component of saliva produced by the salivary glands and of pancreatic secretions produced by the pancreas. To measure enzymatic activity , it is necessary to measure the amount of substrate remaining or the amount of product produced through an enzyme-catalyzed reaction. In the reaction catalyzed by amylase, the product, maltose, can be measured utilizing a colorimetric assay . Colorimetric assays utilize a reagent that changes color in the presence of a compound of interest. The reagent that will be utilized is 3,5- dinitrosalicylic acid ( DNS ) which changes color in the presence of maltose (the product) from yellow to a red/orange. The DNS assay utilizes an oxidation-reduction reaction that occurs between a reducing sugar (like maltose) and DNS. The reducing sugar forms free aldehyde or ketone groups which enables the molecule to reduce the DNS reagent (Figure 4). In this oxidation-reduction process maltose is oxidized into maltonic acid and DNS is reduced to ANSA ( 3-amino-5-nitrosalicylic acid ). Figure 4: Reduction of DNS (yellow) by maltose into ANSA (red/orange)

From Department for Education (April 2014) GCE AS and A level subject content for biology, chemistry, physics and psychology. https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/446829/ A_level_science_subject_content.pdf DNS is yellow in color. As maltose is produced through the hydrolysis of starch by amylase, DNS is reduced by the maltose, forming ANSA which is orange/red in color. The reduction of DNS will be catalyzed by heat in a boiling water bath. More maltose produced results in a darker orange/red color (Figure 5). Figure 5: Reducing sugar concentration indicated by DNS assay From https://youtu.be/NtqsWKRW7N8 Link to video on Reducing sugar by DNS method This a colorimetric assay, which produces a colored product (ANSA) that will be used to measure how much maltose is produced. To measure the amount of a colored product, a spectrophotometer will be utilized. The spectrophotometer allows for the quantitative measurement of light transmitted through a sample. This transmittance can then be used to determine the amount of light absorbed ( absorbance ) by that same sample. Figure 6 shows a single wavelength spectrophotometer, the wavelength is set to a particular frequency of light (in this case 540nm) and the light is then passed through the sample.

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