Biology 3A Laboratory: Enzyme FunctionBiology 3A Laboratory: Enzyme Function Objectives • To be able to list the general characteristics of enzymes. • To study the effects of enzymes on the rate of chemical reactions. • To demonstrate the effect of some environmental conditions on enzymatic reactions such as temperature, pH, substrate concentration, enzyme concentration and inhibitors. Introduction All living organisms require energy in order to sustain the many processes involved in life. The energy for these processes is provided by cellular respiration, a catabolic process that releases energy (exergonic), most often as ATP. It is essential that the chemical reactions involved in cellular respiration occur at a rapid rate and within optimum conditions. Enzymes are critical in this process. Enzymes are biological catalysts that accelerate the multitude of anabolic and catabolic chemical reactions (movement, cellular respiration, digestion, growth, etc.), which occur in living organisms. Many of these reactions are not only accelerated by enzymes, but would not occur to any appreciable extent at body temperature without them. Figure 1. Energy of activation with and without an enzyme. Before any chemical reaction can occur, molecules must obtain enough energy (energy of activation - EA). This energy may be provided when one molecule collides with another or from an external source of energy such as heat. The amount of energy required by chemical reactions varies. The greater the energy barrier, the more energy required to drive the reaction. Enzymes increase reaction rates by lowering the energy of activation (Figure 1). Each enzyme catalyzes a specific chemical reaction. The molecules that enzymes work on are called substrates. Each enzyme has one or a few substrates. Whenever a group of substrates are susceptible to Biology 3A Lab Enzymes Page 1 of 8catalysis by a particular enzyme, those substrates are closely related compounds, demonstrating the specificity of enzymes. This specificity is dependent on the three dimensional shape of the enzyme. The catalytic cycle (Figure 2) for enzymatic reactions begins as the reactants (enzyme and substrate) collide and the substrate fits into the active site (Figure 3) of the enzyme. The substrate and enzyme forms the enzyme-substrate complex held together by temporary bonding (hydrophobic interactions, hydrogen and ionic bonds). It is during the complex formation that the chemical reaction(s) takes place resulting in the product(s). Notice that the enzyme also appears with the product(s) in the equation below (Figure 2). Enzymes emerge essentially unchanged upon completion of the chemical reaction and are capable of further catalysis (reusable). E S ES complex E P Enzyme + Substrate Æ Enzyme-Substrate complex Æ Enzyme + Product(s) Figure 2. Catalytic cycle for enzymatic reactions A: TYROSINASE ACTIVITY The enzyme tyrosinase is naturally found in potatoes. The substrate, pyrochatechol is a clear colorless liquid. The product formed from these hydroxyquinone, which has a yellowish color. The appearance of this color indicates that a reaction has occurred between the enzyme and the substrate as shown in equation 1. The degree of color from light yellow to dark yellow corresponds to the amount of end product produced. To quantify the reaction, we will us a spectrophotometer to measure the absorbance of light at 400 nm (the maximum absorbance for the product). Since absorbance is directly related to concentration, a higher value at an absorbance of 400 nm (A400) indicates more end products are produced. In other words, there was more enzymatic activity. Figure 3. Binding of the substrate at the active site of the enzyme Pyrochatechol + O2 + tyrosinase Æ hydroxyquinone + H2O + tyrosinase (1) (colorless substrate) (colored product) A1 Procedure: Characteristics of the Enzyme Reaction 1. Turn on the spectrophotometers and allow them to warm-up for 10 minutes. 2. Make sure the correct filter is inserted, set the spectrophotometer at 475 nm and calibrate with distilled (DI) water. Remember to recalibrate each time if you change the wavelength. NOTE: When placing tube in the spectrophotometer: • Try not to get fingerprints and smudges on the tubes – wipe off any liquids or prints • Mark numbers of on top of the tube, not where the light is passing • Remove parafilm before placing tube into the spectrophotometer 3. Obtain four tubes and label them on the top 1 – 3 and B. 4. Fill tubes 1 – 3 according to Table 1. Fill the fourth tube, “B” with DI water. This will be the tube to zero or blank the spectrophotometer for the remainder of the lab exercises. 5. Using a small piece of parafilm, cover the top of each tube and invert several times to mix. 6. Note the color and record it on the worksheet. 7. Place the tubes upright in the test tube rack. After 5 minutes determine the A475 and record. Biology 3A Lab Enzymes Page 2 of 8Biology 3A Lab Enzymes Page 3 of 8 CAUTION: Pyrocatechol is a poison! Avoid contact with all solutions. Do not pipette any solutions by mouth. Wash hands thoroughly after each experiment. If a spill occurs, notify the instructor. If the instructor is unavailable, wear disposable gloves and use dry paper towels to wipe up the spill. Follow dry towels with towels soaked in soap and water. Dispose of all towels in the trash. Table 1. Experimental Conditions to Test the Tryosinase Activity Tube DI water Enzyme Substrate Sucrose 1 5 ml 1 ml 1 ml --- 2 6 ml --- 1 ml --- 3 5 ml 1 ml --- 1 ml A2 Procedure: The Effects of Enzyme Concentration 1. Label three test tubes 1 – 3. 2. Fill each tube according the Table 2 (add the substrate last to all tubes). 3. Cover with parafilm and mix by inverting. 4. Note and record the color of each tube. 5. Determine the A475 of each tube after 3 minutes. Table 2. Experimental Conditions to Test the Effects of Enzyme Concentration Tube DI water Enzyme Substrate 1 5.5 ml 0.5 ml 1 ml 2 5 ml 1 ml 1 ml 3 4 ml 2 ml 1 ml A3 Procedure: The Effects of Substrate Concentration 1. Label three test tubes 1 – 3. 2. Fill each tube according the Table 3 (add the enzyme last to all tubes). 3. Cover with parafilm and mix by inverting. 4. Note and record the color of each tube. 5.