Biology

BIOL 102: Lab 6

Enzyme Catalysis

PRE-LAB ASSIGNMENT: Students are expected to read pages 1 to 4 before coming to the lab to complete the experiments.

Print this entire lab packet and bring it to the laboratory.

Please provide a FULL lab report for this experiment following the “Lab Report Guidelines”.

Objectives:

 Observe the reaction of catalase and hydrogen peroxide

 Demonstrate the effects of extreme temperatures on catalase activity

 Learn how to establish a baseline for the amount of peroxide in a 1.5% solution.

 Use titration techniques to determine the rate of hydrogen peroxide decomposition by enzyme catalysis

 Investigate spontaneous decomposition of hydrogen peroxide to oxygen and water.

Background:

Enzyme Structure and Function Thousands of different kinds of chemical reactions take place in a living cell, and nearly all of them are mediated by enzymes. Enzymes are biomolecules that speed up chemical reactions. Without the action of enzymes, metabolic reactions would be extremely slow. For example, if you recall from an earlier unit that carbohydrates are a major source of energy for all cells. They exist in several classes: monosaccharides are simple sugars; disaccharides are composed of two monosaccharides; polysaccharides are made up of three or more monosaccharides. Organisms take in the various carbohydrates as nutrients. Eventually the molecules would break down into monosaccharides on their own, but not in time to support a cell’s life. Therefore, cells utilize enzymes to speed up the process. Enzyme-Substrate Kinetics Enzymes are catalysts, chemicals that speed up chemical reactions without itself being changed in the course of the reaction. Most enzymes are highly specific in their action, catalyzing only one type of chemical reaction or, at most, a small number of very similar reactions. The specific reactants that bind to a particular enzyme are called substrates. Substrates bind to the enzyme at the active site. Active sites only allow selective substrates to bind to it and are responsible for the enzyme-substrate specificity. When the enzyme is reacting with the substrate, a complex is formed.

The figure to the left illustrates the specificity of the substrate to the enzyme and the enzyme-substrate complex that is formed after they bind. Note that in the picture, the arrows go in both directions. This illustrates the principle of reversibility, meaning that an enzyme can break down substrates, as well as put the substrates back together.

In some enzymes, the shape of it is not exactly complementary to the shape of the substrate. The binding of the substrate causes a conformational change that induces a tighter and more accurate fit for itself within the active site and stabilizes the interaction between enzyme and substrate. This is called the “induced fit model.” Did you know? There are over 2,000 known enzymes and each one is involved in a specific chemical reaction.

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Roles of Enzymes In order for molecules to react with each other, the reactants must bump into each other fast enough and in the correct orientation. This set of conditions creates an energy barrier that must be overcome. The minimum energy input that enables molecules to react is called the activation energy (EA). Enzymes lower the energy barrier by bringing substrates close together. Note that an enzyme simply increases the rate of a reaction that could occur spontaneously. The graph illustrates the differences between the amount of activation energy required by an enzyme-catalyzed reaction and an uncatalyzed reaction. Enzyme Regulation Enzymes generally work best under certain narrowly defined conditions such as temperature, pH, and the concentration of the enzyme or substrate. Effect of Temperature Most enzymes have an optimal temperature at which the rate of the reaction is fastest. At a lower temperature, the reaction occurs slowly or not at all. At higher temperatures, the rate of the reaction will increase. As a general rule, an increase of 10⁰C doubles the rate of most chemical reactions including those catalyzed by enzymes. However, enzymes are proteins and high temperatures can denature them. This means that it destroys their tertiary structure and renders the enzyme inoperative. Did you know? Thermolabile enzymes, such as those responsible for the color patterns in Siamese cats and color camouflage of the Arctic fox, work better at lower temperatures. Effect of pH Most enzymes are active over a narrow pH range and have an optimal pH at which the reaction is fastest. The optimal pH for most human enzymes is between 6 and 8. However, there are some enzymes that work best in acidic or basic conditions. The activity of an enzyme may be altered if the pH of its environment is changed. Changes in the pH alters electric charges on the enzyme that may affect the ionic bonds that contribute to its tertiary (and quaternary) structure. A change in the enzyme’s conformation will affect its function. Effect of Concentration Since enzyme reactions are reversible, it could be speculated that the enzymes could be caught in a loop, forming products, breaking them down, and reassembling them, over and over. Yet enzymes follow the Law of Mass Action: the direction taken by an enzyme-catalyzed reaction is directly dependent on the relative concentration of the enzyme, substrate, and product. When there is a great deal of substrate and a little product, the reaction will form more product. Conversely, when there is a great deal of product and little enzyme, the reaction will form more substrate. Did you know? Enzymatic reactions can occur very quickly. For example, carbonic anhydrase causes chemicals within the human body to react ten times faster than without the enzyme present.

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Coupled Reactions Exergonic reactions, where free energy is released, can occur spontaneously, but endergonic reactions require an energy input and thus, cannot proceed spontaneously. Cells will often pair a spontaneous exergonic reaction with a nonspontaneous, energy-requiring reaction. An example of a coupled reaction occurs in the sodium-potassium pump in cell membranes. This exergonic action pumps sodium and potassium against the concentration gradient using ATP and causing an endergonic reaction. Reactions such as these are often seen in cells as the battle to capture and utilize energy occurs. Coenzymes Some enzymes consist only of a protein and function individually. Some enzymes require the use of a coenzyme and they only function when combined. A coenzyme is an organic, non-polypeptide compound that binds to the enzyme. Coenzymes have a varied role. Some are not tightly bound and can move from enzyme to enzyme, transferring electrons or protons. Some alter substrates for a better fit with the enzyme. Still others, bound into membranes, are essential to the energy conversion reactions of photosynthesis and respiration. Many coenzymes must be taken in by animals and are not synthesized; these are collectively referred to as vitamins. Competitive Inhibitors Molecules that bind to the active site of an enzyme and compete with the substrate are called competitive inhibitors. Note that the inhibitor is similar enough to the substrate that it can fit in the active site, but it is not the same as the substrate. While the bonding of the inhibitor produces no product, it blocks the substrate from binding to the enzyme. Competitive inhibition is reversible and behaves the same as an enzyme-substrate complex, with constant binding and unbinding of the complex due to the Law of Mass Action. In competitive inhibition, the active site is occupied by the substrate half of the time and the inhibitor half of the time. If the concentration of the inhibitor is greater than the substrate, the reaction will slow down; otherwise, the inhibitor has little effect. Noncompetitive Inhibitors Unlike competitive inhibitors, noncompetitive inhibitors bind to a region of the enzyme other than the active site and alters the shape so that the substrate cannot bind to the active site. Since there is no competition for the active site, the Law of Mass Action will not come into play in this situation, and a buildup of substrate will not make a difference in accelerating the reaction. Did you know? Many toxic substances like poisons owe their properties to their ability to act as inhibitors to enzymes responsible for catalyzing important biological processes. Examples of these toxic substances include cyanide, heavy metals such as lead, mercury and chromium, and pesticides. Allosteric Controls Some enzymes have an allosteric site, a site other than the active site and when an allosteric regulator (called an effector) binds to it, it causes a conformational change of the active site. In metabolic pathways, enzymes exist in two forms: active and inactive. The active form is rendered inactive by an effector, often a product of an earlier enzymatic reaction. When the products of a metabolic pathway inhibit an earlier step in the pathway, it is referred to as feedback inhibition, or negative feedback. Not all allosteric effectors inhibit an enzyme. In allosteric promotion, the effector actually activates an inactive enzyme. Both of these regulatory mechanisms are extremely effective and can actually work together, allowing the cell to store enzymes in both their active and inactive forms. In allosteric inhibition, if a product down the metabolic line begins to build up, it is not to the cell’s advantage to continue to make it. The product itself will “turn down” the reaction until most of the product has been metabolized. In allosteric promotion, that same product (or a different one) will activate an enzyme to begin reacting with a substrate. Did you know? Allostery means ‘different shape”. Allosteric enzymes change shapes between active and inactive forms as a result of the binding of substrates at the active site and/or regulatory molecules at other sites.

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The Role of Catalase Hydrogen peroxide (H2O2), an antiseptic that effectively destroys cells, is actually spontaneously produced in every cell of a human body as waste. The enzyme catalase, found in peroxisomes, breaks down hydrogen peroxide into water and oxygen gas, preventing it from destroying our body’s cells. Did you know? Hydrogen peroxide is commonly used for washing out cuts or scrapes on the skin. Blood contains catalase, and when the catalase comes in contact with hydrogen peroxide, it turns the H2O2 into H2O and O2. The bubbles and foam that you see are pure oxygen bubbles created by the catalase. Protein Structure Enzymes are made of long chains of amino acids called polypeptides. Although there are only twenty different amino acids, a polypeptide may have hundreds of amino acids repeated in varying sequences. All twenty amino adds have an amine group, a carboxyl group, a hydrogen atom, and an R group, as diagrammed (right). In aqueous solutions, the amine group (NH2) ionizes as a base, gaining a proton. The carboxyl group (COOH) has acidic tendencies, releasing a proton. It is in the structure of the R group that the twenty amino acids differ. Proteins are synthesized on the ribosome; the sequence of amino acids is the primary structure of the protein. Once the polypeptide strand leaves the ribosome, the amino acids begin to interact. The secondary structure involves the formation of helical twists to the protein or, in some cases, a ribbon-like pleated sheet. Once the secondary twists have occurred, the R groups begin to interact, twisting the polypeptide into its final three-dimensional shape. This is called the tertiary structure. In larger enzymes, particularly those showing allostery, a quaternary structure occurs when two or more polypeptides combine. Did you know? Structural Proteomics is the field which scientist attempt to reveal the structure of all the key “functional” sites of any human protein. This will result in the development of highly specific drugs, thus leading to safer and more effective pharmaceuticals.

Some information was adapted from:

Singh-Cundy, Anu and Gary Shin. Discover Biology. 6th ed., Norton, 2015. Solomon, Eldra P. et al. Biology. 10th ed. Cengage, 2015.

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LAB DATASHEET

IMPORTANT: Wear proper protective equipment such as gloves, safety goggles, and a lab apron.

Exercise 1: Testing Enzyme Activity

Materials: 30 ml 1.5% hydrogen peroxide 1 ml catalase working solution 1 ml boiled catalase working solution 1 10-ml syringe 1 1-ml pipette

1 glass rod 1 knife or scalpel 2 2-oz. plastic cups 1 250-ml beaker 1 piece of a potato

Procedure A: Testing Enzymatic Activity 1. Obtain a 10-ml syringe, remove the tip, and label the syringe ‘H’ for hydrogen peroxide. 2. Using the syringe, add 10 ml of hydrogen peroxide to the 2-oz. plastic cup.

(Do NOT dispose of the syringe. You will use it in the next Part 2.) 3. Using a pipette, add 1 ml of catalase solution to the cup. 4. Swirl the contents in the cup to mix the solution. 5. Observe for approximately 30-60 seconds. 6. Record any observations in Table 1. Procedure B: Effect of Extreme Temperature on Enzyme Activity 1. Using the same syringe, dispense 10 ml of hydrogen peroxide solution into another 2-oz. cup. 2. Add 1 ml of the boiled catalase to the 2-oz. plastic cup. 3. Swirl the contents in the cup to mix the solution. 4. Observe for approximately 30-60 seconds. 5. Record any observations in Table 1. Procedure C: Presence of Catalase in Living Tissue

IMPORTANT: Use extreme caution when working with sharp objects. Cut the item on a flat surface to avoid causing any bodily harm. Never cut an item while holding it in your hand.

1. Using a knife or scalpel, carefully cut a 1-cm cube of a potato. 2. Place the tissue in the 50-ml beaker. Mash it with the glass rod. 3. Add 10 ml of hydrogen peroxide to the beaker containing the mashed tissue. 4. Observe any reaction that takes place. 5. Record your observations in Table 1.

Table 1: Enzyme Activity

A. Enzyme Activity

B. Effect of Temperature

C. Presence of Catalase

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Exercise 2: Establishing a Baseline: Determining the Amount of Hydrogen Peroxide in a 1.5% Solution

Materials: 10 ml 1.5% hydrogen peroxide 1 ml catalase working solution 10 ml 1M sulfuric acid

2% potassium permanganate (KMnO4).

1 ml distilled water 2 10-ml syringe 1 1-ml pipette 2 2-oz. plastic cups 1 titration syringe

Procedure: 1. Obtain 2 10-ml syringe, remove the tips, and label one syringe ‘S’ for sulfuric acid and the other ‘T’ for

transfer. 2. Using the correct syringe labeled ‘H’, add 10 ml of hydrogen peroxide to the 2-oz. plastic cup. 3. Using the pipette, add 1 ml of distilled water to the same cup. 4. Using the syringe labeled ‘S’, carefully add 10 ml of sulfuric acid to the cup. 5. Swirl the contents in the cup to mix the solution. 6. Using the syringe labeled ‘T’, transfer 10 ml of the mixture into a new 2-oz. plastic cup. 7. Fill the titration syringe to the 10-ml marking with potassium permanganate. Note the initial reading in

Table 2. 8. Slowly add one drop of potassium permanganate to the plastic cup containing 10-ml of the mixture. 9. Swirl the contents in the cup to mix the solution. 10. Continue to add potassium permanganate, one drop at a time and swirl after each addition, until the

solution permanently turns pink or brown. The amount of KMnO4 added is proportional to the amount of H2O2 that was present in the solution. If you use all of the potassium permanganate in the syringe, refill to the 10-ml mark and continue your titration.

11. Record the final volume in the titration syringe in Table 2.

Table 2: Establishing a Baseline

Initial Reading of Titration Syringe

Final Reading of Titration Syringe

Baseline (Initial – Final)

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