Effects The of the Enzyme-Substrate Complex, Competitive Inhibition, and Denaturation of the Enzyme on Enzyme Reactions
Shanna Moore Student ID #00608051 Section A21 February 10, 2007
Abstract The purpose of this experiment was to examine the effects of a substrate and inhibitor, ad well as the effects of denaturation on an enzyme. Since enzymes are essential for even the most basic functions of life, it is important to understand how they work, and what they need to create a reaction. Qualitative observations were made on the color change of each test tube in this experiment. With the help of controlled variables, it was possible to determine what effects a lack of substrate, presence of substrate, presence of inhibitor, and denaturation of the enzyme had on the enzyme. It was determined that it is necessary to have a substrate present for a reaction to occur, while the presence of an inhibitor and denaturation of the enzyme prevents a reaction from occurring.
Introduction Enzymes are proteins that facilitate the basic chemical reactions that facilitate life. This experiment examines the effects of certain amino acids on an enzyme. The basic question being, what happens when the enzyme is combined with certain things such as a substrate or inhibitor? Qualitative observations were made to determine which reactions occurred and which did not. These were made by examining the color change in each of the four test tubes. Enzymes require a substrate in order for a reaction to occur. Enzymesubstrate combinations when in the presence of an inhibitor, in this experiment Malonate, will not be able to sufficiently catalyze the enzyme. Enzymes will not produce a reaction when denaturation occurs either. While utilizing two controls, this experiment will examine the effects of the substrate, inhibitor, and denaturation on the enzyme, and its ability to produce a reaction. It was hypothesized that test tube #1 would not change colors, because the enzyme was not combined with a substrate. The goal of this
experiment is to demonstrate how the substrate and inhibitor act on the enzyme to produce or inhibit specific reactions.
Methods and Materials Four test tubes were obtained and labeled 1, 2, 3, and 4. Ground beef was previously prepared by the instructor. A pea sized piece of the meat was placed in each of the first three test tubes. The following number of drops of Malonate, Succinate, Phosphate Buffer, Water, and Methylene Blue were added to the test tubes.
Contents of Test Tubes Test Tube Enzyme (in meat) Malonate (inhibitor) Succinate (substrate) Phosphate buffer Water Methylene blue Total drops 1 Fresh 0 0 12 10 3 25 2 Fresh 0 4 12 6 3 25 3 Fresh 4 4 12 2 3 25 4 Boiled 0 4 12 6 3 25
Finally, the test tubes were sealed with paraffin oil. This preserves the reactions while preventing outside contamination. The tubes were placed in a 37 C water bath and allowed to incubate for 30 minutes. After the 30 minutes, the tubes were swirled around
and allowed to incubate for another 10 minutes. The observations were then made and results were recorded.
Results and Discussion The results for the experiment were as follows. Test tube #1 showed no clear color change. Test tube #2 showed a very significant color change, with the contents of the test tube changing from a dark blue to a clear solution. Test tube # 3 showed a slight, but noticeable color change from dark blue to light blue. Test tube #4 showed no clear color change. The results of the following experiment are represented by the table below (Figure 1). Figure 1: Qualitative Observations of Color Change
Sample Number
Change Observed
Test Tube #1
No Change
Test Tube #2
Color change from blue to clear.
Test Tube #3
Slight color change from dark blue to light blue No Change
Test Tube #4
The first test tube is testing what happens when the enzyme is not combined with either the inhibitor or the substrate. Since there was no change, it shows that without combination, the
substrate no reaction will occur. This is because the substrate combines with the enzyme and acts as a key that allows the reaction to go through. This is illustrated by the following diagram (Kimball, 1994).
Test tube two was one of the controls. It contained the Succinate substrate. The reaction completely occurred in this reaction because the substrate is the key that allows the reaction to occur. The test tube completely changed color from a dark blue to a clear color. This was a control because it is important to see what a completely reacted test tube would look like when comparing each test tube to see if a reaction occurred. Test tube three was the other control. This reaction demonstrates the effects of competitive inhibition. Due to the presence of the inhibitor, the reaction is blocked. The inhibitor binds to the enzyme an blocks a reaction from occurring. This is called competitive inhibition because increasing the ratio can gradually restore catalysis rate (Kimball, 2004). The final test tube, the one the contained the boiled meat sample. This reaction demonstrates the effects of denaturation on the enzyme. A reaction did not occur in this experiment because the denaturation of the enzyme prevented the substrate-enzyme reaction from occurring. Overall the reactions produced from this experiment followed the expected results. The enzyme when combined with the substrate produced a complete reaction. The enzyme when combined with the inhibitor showed a slight reaction, yet it as clear
that the reaction was inhibited. The enzyme when denaturation occurred (the boiled enzyme) did not produce a reaction at all, and the enzyme when was not combined with the substrate also did not produce a reaction. The hypothesis was supported by the results of this experiment. In the absence of the substrate, no reaction occurred in test tube A. This was demonstrated by the fact that throughout the experiment, test tube A maintained its dark blue color, thus indicating that no reaction occurred. This was compared with the test tube that contained the substrate, where a clear reaction occurred demonstrated by the color change from dark blue to clear. There were a few possible sources of error for this experiment. First, it is possible that the exact number of drops placed in each test tube were slightly off of the suggested numbers. This is due to the fact that a few of the droppers were more sensitive than others. This is specifically important in the test tube demonstrating competitive inhibition, because if there is more substrate than inhibitor the ratio could be such that the substrate could overcome the inhibition and a reaction could still occur. The second possible source of error had to do with the inexact pouring of the paraffin oil. In one of the test tubes more than the recommended amount got poured into the tube. While this did not appear to affect the results, it is possible that it could have some adverse effect on the experiment.
Conclusion There are a few major conclusions that can be made from this experiment. The first is that then enzyme-substrate combination is a necessity for the reaction to occur. The next major conclusion is that, when in the presence of an inhibitor, a reaction is
unable to occur, or is severely restricted. This can be overcome by increasing the ratio or substrate to inhibitor, but overall it is difficult to get a reaction to occur in the presence of an inhibitor. Finally, when denaturation of the enzyme occurs a substrate is not able to combine with the enzyme to form a reaction.
References Campbell, Neil A. and Reece, Jane B. Biology. 7th edition. Pearson Benjamin Cummings. Frankel, Joseph, et al. (2005). Some Basic Biochemistry. Principles of Biology I Laboratory Manual. 5th edition. Pearson Custom Publishing. pp 29-36. Kimball, John W. Enzymes. (December 9, 2004). Available at: http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Enzymes.html