Competitive Inhibition

Introduction

Enzymes are protein catalysts that promote and regulate chemical reactions within cells. Enzymes are not used up in the reactions that they aid and can be used to catalyze a second set of substrates once it completes the reaction for a first set of substrates. There are many enzymes present in all cells, each with a specific structure and function. The composition of a protein determines its structure and its function are based on its structure. Enzymes as well as other proteins have active sites that allow a specific type of molecule to bind to the site. When a substrate or inhibitor binds to the active site, the structure of the enzyme may change including other active sites on the protein. Substrates are the reactants of which are to be made into products with facilitation by the enzyme. Inhibitors, on the other hand, can be of two types. Competitive inhibitors bind to an enzyme's active site in place of a substrate. These inhibitors compete for the substrate's active site and prevent the enzyme from working by directly blocking the enzyme's contact with substrates. This returns the substrate's reaction rate to the uncatalyzed rate, which is much slower than when the catalyst enzyme is present. The other type of enzyme inhibitor binds to a site different from that of the substrate's active site. When these non-competitive inhibitors bind, they change the shape of the substrate's active site and the substrate is unable to bind to the changed active site. This also returns the substrate's reaction rate to the slower, uncatalyzed rate.

Inhibitors are not the only factors that determine an enzyme's efficiency. All enzymes have optimum operating conditions that include temperature, pH, and substrate concentration. If the temperature becomes too high, enzymes loose their natural structure and function and are termed denatured. Enzymes also depend on pH concentration for their ability to function. Too acidic or too basic an environment, the enzyme will assume a different shape and will not be able to catalyze the conversion of substrates into products. It also is logical that increased substrate concentration will increase the chances of a substrate particle coming into contact with the enzyme and reacting to form products.

Abstract

The purpose of this experiment was to test our hypothesis about enzyme activity under different conditions. Succinic Dehydrogenase, which is an important enzyme used in cellular respiration, was chosen for examination. Succinic dehydrogenase is found in mitochondria, which are in abundant in muscle cells. This being the case, beef heart was chosen for the source. The reaction that succinic dehydrogenase facilitates is shown below.

Succinic Dehydrogenase

|

COOH-CH2-CH2-COOH + FAD→ COOH-CH=CH-COOH + FADH2

| |

Succinic Acid Fumaric Acid

It has been found that methylene blue can be used for a hydrogen acceptor instead of FAD. Reduced methylene blue is clear in color and will be used in our experiment to reveal, relatively, how much succinate has been converted to fumaric acid. The clearer the resulting solution, the more the reaction has progressed. Methylene blue in this case is being used as an indicator. We will be using malonic acid for our competitive inhibitor. Malonic acid can bind to succinic dehydrogenase because it is structurally similar to succinic acid. The malonic acid blocks succinic acid molecules from binding to active sites by occupying the active site meant for succinic acid. Since the reaction is not completed, the enzyme remains attached to the inhibitor for a much longer time than it would for a substrate since it will not form a product. The inhibitor will take longer to be released by the enzyme than its normal manner after the product is formed.

For this experiment we will use four tubes with equal total volumes of liquid, equal volumes of phosphate buffer (to keep pH constant due to different concentrations of acids in solution), and equal volumes of methylene blue. The variables to be tested will be the presence of substrate, presence of an inhibitor, and activity of a denatured enzyme.

Hypothesis

Test tube one should stay blue because there is no substrate for the succinic dehydrogenase to act upon, thus not being able to reduce the methylene blue, and the methylene blue should not change color.

Test tube two should be the most clear because the enzyme is not denatured, there are no inhibitors present, and there is substrate present. The reaction should proceed to completion, and methyl blue would be reduced by the presence of the reaction, making it clearer in color.

Test tube three may change to a slightly less dark blue than it was initially. This is because there is an inhibitor present which will block some of the enzyme molecules from binding to succinic acid. Some succinic dehydrogenase molecules may remain uninhibited if the concentration of the inhibitor was not high enough. These few uninhibited enzymes will still be able to aid the reaction. This will be a small fraction, so a little of the methyl blue will be reduced and become clear, giving a slightly lighter overall shade of blue.

Test tube four should not change in color enough to be visible to the eye. Test tube four contains no natured enzyme because it has been boiled and denatured, meaning it neither retains its structure or function. The succinic dehydrogenase will be unable to accept succinic acid because overheating drastically changed the active site's shape. Some of the succinate molecules may naturally form fumaric acid, but at a very slow rate, which would cause the reduction of the methylene blue and therefore the color would be very slightly less blue, but this would not be due to enzyme activity. Since the reaction would not occur, methylene blue will not be reduced and there will be no color change

Procedure

To obtain the necessary succinic dehydrogenase, which is found in abundance in mitochondria, we used a fifteen-gram piece of frozen beef heart slightly thawed. Our TA finely minced the beef heart piece with a razor and added cold phosphate buffer (enough to make pasty) to the meat and ground the meat with a mortar and pestle for three minutes. The meat was then washed with more cold phosphate buffer and strained through a double layer of cheesecloth. This process was repeated a few times with the strained

...