Enzymes: A Quantitative Approach
ENZYMES ARE ESSENTIAL FOR LIFE
Not all chemical reactions occur at the rapid rates we observe in lab settings. Many of these slow reactions, such as some metabolic processes, are essential for an organism to survive but, paradoxically, are not quick enough to sustain life. Biological catalysts are chemical agents that influence the rate of a reaction without changing the reaction. An enzyme is a catalytic protein that allows reactions to occur at much higher rates. With the help of enzymes, those slow reactions can occur quickly enough to sustain life. The amount of energy needed to begin the bond-breaking process in a reaction is known as the activation energy. Enzymes speed up reactions by lowering this energy barrier therefore allowing reactions to proceed much earlier than they would have with a higher energy barrier.
ENZYME-SUBSTRATE COMPLEX
Enzymes are substrate-specific. This means they are very “picky” and only react with specific substrates to form products. For example, this week in lab you will work with the enzyme, tyrosinase. Tyrosinase is found in many organisms, including humans. This lab will focus on the role tyrosinase plays in potatoes. (Note the suffix –ase in tyrosinase; this suffix nearly always denotes an enzyme.) Tyrosinase reacts with its substrate pyrocatechol to create the plant antibiotic hydroxyquinone. Hydroxyquinone is a yellowish-brown liquid that is found at the site of plant injury. Think of a bruise on your apple. The brown color is hydroxyquinone. In a normal enzymatic reaction the enzyme and substrate collide randomly in a solution and join at the enzyme’s active site. This collision works much like a lock and key effect. The active site has a specific shape that only a particular substrate fits into (with the exception of inhibitors which we will discuss later on). When the two are joined they react and the substrate is converted into a product (in our experiment, hydroxyquinone). Once the reaction is complete the enzyme and newly formed product separate with the enzyme left unchanged. Below is an example of the enzymatic reaction between the sugar, sucrose (yellow disaccharide), and its enzyme, sucrase (purple protein). In the end, sucrose is converted to glucose (orange monosaccharide) and fructose (pink monosaccharide).
ENVIRONMENT AFFECTS ENZYMATIC ACTIVITY
This week’s lab will explore what happens to an enzymatic reaction under different environmental reactions. You will test the reaction between tyrosinase and pyrocatechol under normal conditions (your control) and under 5 environmental variations: 1) an alternate substrate, 2) variable concentration, 3) variable temperature regimes, 4) variable pH levels, and 5) in the presence of an inhibitor. Let us explore what happens when we change the conditions under which the enzymatic reaction occurs.
Control Reaction
As explained previously the reaction between the enzyme, tyrosinase and its substrate, pyrocatechol creates the product, hydroxyquinone. Hydroxyquinone is a yellowish-brown color. A positive reaction should yield a yellow-brown liquid indicating the creation of hydroxyquinone. A negative reaction will remain the clear-pink color of tyrosinase. Your positive control was the reaction between tyrosinase and pyrocatechol under normal conditions. Your negative controls were those with tyrosinase or pyrocatechol combined with only water. Obviously no reaction will take place without both enzyme and substrate present.
An Alternative Substrate
As mentioned previously, enzymes are substrate-specific. You will test substrate-specificity by combining the enzyme, tyrosinase with an alternate substrate, sucrose. If enzymes are indeed substrate-specific you should expect no reaction between the two molecules and a negative test should result. Did you obtain these results?
Variable Concentration Regime
The reaction between an enzyme and its substrate is entirely random. They do not seek one another rather they collide randomly in solution. If the concentration of one molecule or another is increased, the chance of collision increases as well. In lab you will alter the concentration of enzyme in your experiments. If the concentration increases, what do you expect will happen to the rate and intensity of the overall reaction?
Variable Temperature Regime
Enzymes are proteins and are therefore very sensitive to changes in temperature. As you learned in the Biological Macromolecules lab, protein function is determined by its 3-D shape. If this shape is altered a protein becomes useless. The easiest way to change the 3-D shape of a protein (in this case, enzyme) is to heat it. At high temperatures (the actual temperature depends on the enzyme) the protein will denature, or lose its 3-D shape, and no longer function. Temperature can also affect a reaction by increasing or decreasing the rate of collision between enzyme and substrate. Recall the concept of entropy or increasing randomness from the Biological Membranes lab. If heat is increased than molecules (in this case enzyme and substrate) will move more quickly in solution, collide at higher rates and react more quickly. Conversely, if the temperature is decreased than those same molecules will move more slowly, collide less frequently and therefore, react more slowly. In this lab you will subject the tyrosinase enzymatic reaction to three temperature regimes: heat, room temperature, and cold. Using the above information, one would expect that with increasing heat (and therefore increasing entropy) the enzymatic reaction rate will also increase, BUT, only to a certain point. If the temperature is too high, than the enzyme will denature and the reaction will not run. Did you find these results in your experiment? What happened when the reaction was exposed to cold? Remember, you can measure the rate of this reaction by the speed at which hydroxyquinone is created.
Variable pH Regime
As proteins, enzymes are not only sensitive to temperature but also to the pH (relative acidity or alkalinity) of a solution. Some enzymes react better under neutral pH conditions (pH 7), others in acidic conditions (<pH 7) and still others in basic conditions (>pH 7). In this lab you will test the tyrosinase reaction in pH 5, pH 7, and pH 9. As tyrosinase is an enzyme associated with fruits and vegetables which pH regime would you expect it to work optimally? (Hint: fruits tend to be more acidic) Like the temperature experiment, you can test optimality of the reaction by the speed in which hydroxyquinone is created as well as the intensity of the reaction color.
Presence of an Inhibitor
Inhibitors are molecules that can interfere in an enzymatic reaction. There are two kinds of inhibitors: competitive and non-competitive. A competitive inhibitor binds to the active site of an enzyme, preventing the substrate from reaching the active site and reacting. A non-competitive inhibitor is a little more “sneaky” (this is in quotes because all of these reactions are random as the molecules collide in solution) and binds to a place on the enzyme other than the active site. Once it binds on a remote area, it actually changes the physical configuration of the active site so the substrate no longer fits and the reaction can no longer occur. In this lab you will add an inhibitor, phenylthiourea, to the reaction between tyrosinase and pyrocatechol. We expect that phenylthiourea should prevent the reaction from occurring. Did you obtain these results?
Review Questions
- What are enzymes? How do they affect reactions?
- What was the source of our enzyme? What is its function in nature?
- T/F Enzymes are lipids
- T/F Science seeks to disprove a null or no change hypothesis.
- T/F High temperatures will increase the collision rates of molecules.
- A molecule that binds with an enzyme at the active site and prevents the substrate from binding is called _____________.
- Give 3 different characteristics that describe an enzyme.
- What does it mean when an enzyme is said to be "denatured?"
- What does "saturation" mean?
- How does changing the temperature of an environment affect an enzyme?
- How does enzyme concentration affect reaction rate?
- How does substrate concentration affect reaction rate?
- Why do organisms have narrow ranges of tolerance for changes in pH or temperature in their environment?
- Define competitive inhibitor and non-competitive inhibitor.
- T/F Enzymes do not cause chemical reactions to occur.
- T/F Enzymes are altered in a chemical reaction.
- A substrate and enzyme will bind at what site?
- Enzymes are what type of macromolecule?
- Why are only a small number of enzyme molecules necessary to catalyze a large number of substrate molecules?
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