Group Enzyme Report

The Effects of pH on Enzyme efficiency

Introduction: An enzyme is commonly referred to as a biological catalyst, due to its ability to increase the rate of a metabolic reaction by decreasing or lowering the activation energy (Commonly abbreviated EA) required to perform the reaction. By definition from (Campbell, Reece, Meyers 2008 edition) an enzyme is a macromolecule that acts as a catalyst, a chemical agent that speeds up a reaction without being consumed by the reaction. Therefore enabling the reactant molecules to absorb enough energy to reach the transition state even at moderate temperatures. An enzyme cannot change the G for a reaction; it cannot make an endergonic reaction exergonic. Thus Enzymes can only hasten reactions that would occur eventually anyway, but this function makes it possible for the cell to have a dynamic metabolism, routing chemicals smoothly through the cells metabolic pathways, and because enzymes are very specific for the reactions they catalyze, they determine which chemical processes will be going on in the cell at any particular time. Specifically the way in which an enzyme will go about reducing the activation energy required to initiate the reaction can occur in various ways such as increasing localized pH levels to facilitate H+ transfer or the enzymes active site brings the specific substrate into more applicable positions before the reaction takes place. Enzymes are typically very specific as to which reactions they catalyze and the substrates that are subsequently associated in these reactions. Complementary shape, charge, hydrophilic and hydrophobic characteristics of enzymes and substrates are ultimately accountable for this specificity. In this experiment we shall examine and investigate the enzyme catalase. Catalase is one of the most common enzymes present in all living organisms exposed to 02. This enzyme in particular is responsible for the breakdown of H202 to produce water and oxygen. Because H202 is a harmful waste product produced by many normal metabolic activities, thus to prevent any further damage to the cell it is vital that it is quickly converted into less harmful products. Catalase usually resides in an organelle known as peroxisome, which is present in virtually all-eukaryotic cells. Just as each enzyme has an optimal temperature, it also has a pH at which it is most active. The optimal pH values for most enzymes fall in the range of about 6-8, but there are exceptions of coarse. For example, pepsin, a digestive enzyme in the human stomach, works best at a pH of around 2. Such an acidic environment denatures most enzymes, but pepsin is adapted to maintain its functional three-dimensional structure in the acidic environment of the stomach. In contrast, trypsin, a digestive enzyme also, residing in the alkaline environment of the human intestine has an optimal pH of 8 and would subsequently be denatured in the stomach (Campbell, Reece, Meyers 2008 edition). The pH of a solution can have several differing effects on the structure and overall activity of enzymes. For instance, pH can essentially have an effect of the state of ionization of acidic or basic amino acids. We know that Acidic amino acids have carboxyl functional groups in their side chains and basic amino acids have amine functional groups in their side chains so If the state of ionization of amino acids in a protein is altered then the ionic bonds that help to determine the 3-D shape of the protein can be altered significantly as well. This can lead to altered protein recognition or an enzyme might become denatured. Changes in pH ultimately affect not only the shape of an enzyme but also a change in the shape or charge properties of the substrate so that either the substrate cannot physically bind to the active site or fundamentally it cannot undergo catalysis. From the information just discussed, it is the aim of this experiment to distinguish the optimal pH range for enzyme catalysis. Hypothesis (H1) = The enzyme Catalase, when isolated, will have an optimal pH range between 6-8 and that enzyme activity will essentially decrease when the pH diverges from the optimal range between 6-8.

Group Enzyme Report

If our hypothesis proves to be correct, this will demonstrate that this particular enzyme, catalase, found in almost every living organism exposed to O2 will perform most efficiently at pH levels between 6-8. However we do know that many other factors contribute to optimal enzyme activity, such as temperature, substrate concentration and enzyme concentration. Therefore many variables need to be kept constant to achieve maximum accuracy and reliability. Thus the independent variable of this experiment is the pH level and the dependent variable is the bubbling or effervescence of the reaction vessel. The degree of effervescence or bubbling is a good indication as to how effective the enzyme has performed its role. This experimental design also incorporates the use of multiple control groups according to each source of where the enzyme catalase was originally from, in which just the enzyme catalase, the hydrogen peroxide and 3cm of distilled water are present to essentially gather and observe the full comparisons between the direct impact of adding acidic and basic solutions to the reaction vessel compared to the differing degree of effervescence associated with the source of the catalase, because different sources very well may contain different levels of catalase. Safety Precautions: Take relevant precautions when using such chemicals as: HCL- Corrosive 8 - Avoid contact with skin and eyes. If skin contact occurs wash immediately with water for 15 minutes and seek medical attention. NaOH- Corrosive 8 - Avoid contact with skin and eyes. If skin contact occurs wash immediately with water for 15 minutes and seek medical attention. Similarly take necessary precautions when using and handling any form of biological substances: Always wear gloves Do not digest or consume potatoes, carrots, wheat sprouts or beef liver Always abide by laboratory rules such as no drinking or eating during the lab Wear safety goggles & gloves when need be Materials & Equipment Universal indicator 1M NaOH HCl (0.1-2%) Distilled water Test tubes Hydrogen peroxide Razor blade Scalpel Mortar and pestle Test tube rack Measuring cylinder Potato Liver Carrot Wheat Sprouts Ruler

Group Enzyme Report

Method 1.Take 12 test tubes & set them up in a test tube rack, then mark the test tubes accordingly, 3 X beef, 3 X wheat sprouts, 3 X potato, 3 X carrot and a line at 3cm from the bottom and at 8cm as well on all test tubes, so we can accurately measure how much solutions are in each test tube. Secondly add 3mL of the hydrogen peroxide solution to each. 2.Place 5mL of (0.1 2.0%) HCl into 4 of the test tubes, 5mL of distilled water into another 4 and to the last 4 test tubes add 5mL of 1 M NaOH solution. 3.To each of the 12 test tubes add a couple of drops of universal indicator. 4.Cut three 1cm3 cubes of potato, carrot, wheat sprouts and beef liver. 5.Take a single cube and crush it with the mortar and pestle before adding it to the subsequent test tube. You may wish to use a spatula to transfer the reactant. 6.Observe and measure the amount of effervescence or bubbling that occurs in each test tube. 7. Use pH paper chart to measure the pH of each test tube and record your measurements in a raw data table. 8. Repeat this experiment another three times to attain greater levels of consistency in results to essentially improve reliability and accuracy in observations so we can make clear and concise conclusions in relation to the results of our experiment. Test Tube Set Up

Group Enzyme Report

Expected Results:

Group Enzyme Report

We expect that the enzyme catalase will be generally most active at around a pH of 6-8, and thus in this environment the enzyme should perform at its optimum or peak level and that we should observe the greatest amount of effervescence indicating that the reaction is taking place, converting

Group Enzyme Report

the harmful substance of hydrogen peroxide into less harmful constituents of water and oxygen. This investigation thus models cellular processes carried out in the human body and overall validating our investigation as a model.