The Effect of low pH on Enzyme Activity
Design an experiment in which you will test the effect of an acidic fluid on enzymatic activity. Recall: enzymes are proteins! To complete this project, it may be useful for you to first review the Scientific Method Tutorial, information about pH and enzymes in the text book and course modules, Lab 1 (Introduction to Science) and Lab 4 (Enzymes). As you review Lab 4, you will be reminded that there are several factors that impact enzymatic activity: pH, temperature, and amount of reagent. It is OK to use the same enzyme/substrate/method as you did in lab 4 (but modify the treatment), or you can search on-line to find a different enzyme/substrate/method for measuring enzyme activity for your project (include all references).
As you design your experiment for this project, please remember that you are trying to examine how an acidic fluid will modify the outcome of an enzymatic reaction. To successfully complete this project, you will need to identify the question(s) being asked in your experiment and the hypothesis that you are testing. In your experimental design, you must clearly explain what you are doing. That means that you will need to identify the enzyme, the substrate, the acidic fluid used as treatment, the control treatment and the method of measuring enzyme activity, as well as explain your experimental protocol. You must also thoroughly explain how the acidic fluid impacted enzyme activity based on the results from your own experiment as well as knowledge of enzymes and pH from the text book, modules, lab manual and potentially additional information sources.
Hint: Keep in mind that the acid will change the environmental conditions of the experiment (for example, a low pH value could change the shape of the active site on the enzyme protein), without directly participating in the reaction.
Outline:
Include the following in your outline:
Name of enzyme you will use
Name of organism (if applicable)
The substrate and products in the chemical reaction
Method for measuring enzyme activity
Treatment: acidic fluid(s), pH, length of exposure, how you will treat your samples
The control(s) in the experiment
Hypothesis
How you will present your data (table and/or type of graph)
Anything else you would like to get feedback on before you start your experiment
Write a lab report that includes the following:
1. Title page: descriptive title, your name, course name, semester
2. Introduction: general background information about enzymes and specific information about your chosen enzyme, the question(s) that you are asking and a clear hypothesis for your experiment (20 points).
3. Design an experiment. Provide a detailed description of the materials and methods used to conduct the experiment. Identify control and experimental samples, as well as independent and dependent variables. Also include the methods used for data collection and analysis (20 points).
4. Conduct the experiment and record your results. Take picture(s) of your results. What did you observe? Present your data in table and/or graph format. Remember to label everything and include the unit of measure with all numbers (20 points)
5. Use your knowledge of enzymes and pH to interpret and discuss your results. It may be necessary for you to refer to the OLI course modules, lab manual and/or use additional information resources. What effect does the acidic treatment have on enzyme activity? Did you get the expected results? Explain. (20 points)
Solved
THE EFFECT OF LOW PH ON ENZYME ACTIVITY
Enzymes are biological catalysts which accelerate the rate of biochemical reactions to a rate which can sustain life. Like all catalysts, enzymes work by lowering the activation energy required to initiate the reaction. They, however, do not get used up in the reaction. Enzymes act on biomolecules known as substrates, and the result is different products. There is, however, a distinct difference between enzymes and other catalysts in that they show high substrate specific in that they will only catalyze a reaction involving one substrate and no other. Most enzymes are protein in nature with a three-dimensional structure, their catalytic activity depending on the integrity of their innate protein conformation. When denatured to its constituent subunits, an enzyme’s catalytic activity is lost. Other enzymes consist of catalytic RNA molecules and are thus referred to as ribozymes. Enzymes utilize intermolecular forces thus bringing substrates together in the most optimal way thus facilitating the reaction (Stryer, 2002)
Enzymes possess active sites which facilitate catalysis. Within these active sites are residues which are directly involved in the making and breaking of intermolecular bonds. Enzymes active sites are a three-dimensional cleft consisting of an amino acid sequence. The catalytic activity of some enzymes is solely dependent on their amino acid residue while other enzymes require additional chemical components which can either be inorganic ions such as Ca+, Fe+, or metalloorganic molecules referred to as coenzymes(Stryer et al., 2002)
Like all other chemical reactions, the rate of enzyme-catalyzed reactions is affected by various factors. Some factors that may affect the rate of these reactions include substrate concentration and enzyme inhibitors. Since enzymes are protein in nature, reaction rates may be further affected by temperature and pH (Cox et al., 2013).
Each enzyme has a specific pH and temperature range within which the reaction is at optimum. When an enzyme is subjected to extremely high or low pH or temperature, it may lead to irreversible protein denaturation thus stopping the reaction (Stryer, 2002).
Amylase is an enzyme involved in the hydrolysis of starch to constituent sugars. The enzyme is present in the saliva of humans as well as other mammals thus initiating the process of carbohydrate digestion in the mouth. Amylase can be classified into three categories namely α-Amylase, β-Amylase, and γ-Amylase. Amylase hydrolyzes the α-1, 4-glycosidic bonds of starch to yield disaccharides. This explains why foods rich in starch achieve a sweet taste when chewed (Ramasubbu et al., 1996).
Like all other enzymes, amylase has a pH within which catalysis is at optimum. When subjected to a pH below or above optimum pH, the rate of starch hydrolysis reduces. Extremely high and low pH leads to irreversible denaturation of the enzyme thus stopping the reaction.
α-Amylase, a calcium metalloenzyme is a major digestive enzyme and can be found in the saliva and pancreas. It can also be found in plants and bacteria (Ramasubbu et al, 1996). The experiment conducted uses saliva which contains salivary amylase to determine the effect of low pH on amylase by determining the rate of starch hydrolysis under different pH ranges.
Hypothesis
- Low pH has a negative effect on the enzymatic activity of amylase. A lower reaction rate will, therefore, be observed.
Reagents & Material.
- Test tubes
- pH buffers; pH 3, pH 5, pH 7
- Iodine solution
- Starch solution
- Amylase
- Water baths at 370C
- Distilled water
Procedure
- To each of the three test tubes, 5ml of starch was added
- In the first test tube, 2ml of pH three buffer was added
- To the second test tube, 2ml of pH 5buffer was added
- In the third test tube, 2ml of pH seven buffer was added
- To each of the three test tubes, 1ml of amylase was added
- The tubes were incubated at 370 C
- The time the reaction took and the color change was noted. This was used to determine the reaction rate for each of the test tubes
Results
The results obtained are as tabulated below
| Tube label |
1 |
2 |
3 (control) |
| pH |
3 |
5 |
7 |
| Time (min) |
2 |
1.1 |
0.8 |
| Starch conc. (mg/ml) |
0.037 |
0.012 |
0.030 |
| Reaction rate (mg/ml per minute) |
0.00550 |
0.037 |
0.0017 |
The results obtained confirm that the pH of the medium does indeed affect the hydrolysis of starch by amylase. The reaction rate at pH 3 is considerably low. The reaction increases as the pH increase with pH seven recording the highest reaction rate.
Discussion
The results obtained show a direct impact of the pH concentration and the rate of an enzyme-catalyzed reaction. As described, most enzymes are protein n nature with their catalysis being dependent on their native amino acid conformation on its active sites. Enzymes interact with the substrate through these active sites catalyzing the reaction thus forming constituent products after which the enzymes detaches and moves to interact with other substrate molecules. Interference with an enzyme’s active site could be therefore either reversible or irreversibly interferes with its ability to catalyze biochemical reactions (Stryer et al., 2002).
Several factors may affect an enzyme’s active sites. One of the factors is enzyme inhibitors. Inhibitors could either be competitive or non-competitive. Competitive inhibitors will reversibly bind to an enzyme leading to a temporary loss in enzymes catalytic ability which non-competitive enzyme inhibitors bind irreversibly to an enzyme thus leading to permanent loss of an enzyme’s catalytic ability. The effects of non-competitive enzyme inhibitors could be dire and lead to the inhibition of major biochemical and metabolic pathways which could ultimately lead to the death of organisms. It is through non-competitive enzyme inhibition that many poisons that poison such as pesticide and insecticide have been developed (Stryer et al., 2002).
Temperate and pH also directly affect an enzyme’s active sites and hence enzymatic activity. With most enzymes being protein in nature, their functionality is dependent on their secondary and tertiary structures. Exposure to low temperature could lead to inactivation or denaturation while exposure to extremely high temperature could break the bond involved in the formation of the enzyme’s secondary structures. This eventually leads to irreversible enzyme denaturation and permanent loss of enzymatic activity (Stryer, 2002).
Hydrogen ion concentration also termed as pH also affects enzyme activity the same way temperature does. The medium in which an enzyme is subjected to should not interfere with, and enzyme’s structural integrity as this affects tan enzyme’s catalytic activity. The results obtained show that amylase shows optimum enzyme activity within a neutral pH range with its enzyme activity deducing with a reduction in pH. While most enzymes show optimum enzyme activity at neutral pH, it is important to note that different enzymes have different pH requirements. Some enzymes such as the digestive enzymes such as pepsin thrive at a very low pH of about pH 2 while other enzymes show optimum activity at basic pH (Stryer, 2002).
An enzyme’s optimum pH and temperature are however mostly dictated by the organism’s habitat.
Conclusion
Enzymes play a crucial role in the survival of organisms. By accelerating the rate of reactions some of which would take years, enzymes can sustain life. With most enzymes being protein in nature, they possess primary, secondary and tertiary amino acid structures. Their catalytic activity is dependent on the integrity of the secondary and tertiary structure of their active sites. The integrity of these structures is affected by various factors including temperature and pH. At optimum pH and temperature, the rate of the reaction proceeds at an optimum rate. Extreme temperature and pH could however reversibly or irreversibly interfere with enzymatic activity either slowing down the reaction rate or denaturing the enzyme thus stopping the reaction completely. Each enzyme has a pH within which the reaction rate is fastest. While some enzymes prefer a neutral pH, others opt for an acidic or basic pH. It is therefore important to research on an enzyme is conducted to determine the conditions under which enzymatic activity is at optimum.
References
Stryer L, Berg JM, Tymoczko JL (2002). Biochemistry (5th Ed.). San Francisco: W.H. Freeman
Cox MM, Nelson DL (2013). “Chapter 6.2: How enzymes work”. Lehninger Principles of Biochemistry (6th Ed.).New York, N.Y.: W.H. Freeman.
Ramasubbu, N.; Paloth, V.; Luo, Y.; Brayer, G. D.; Levine, M. J. (1996). “Structure of Human Salivary α-Amylase at 1.6 Å Resolution: Implications for its Role in the Oral Cavity”.
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