Effect of Concentration and Shape on Enzyme Function
Chemically, enzymes are proteins. Each specific enzyme has a unique physical structure that is essential for
its function. The shape of each specific enzyme "fits" the shape of the reacting molecule(s) for which the
enzyme serves as a catalyst. Because of the enzyme "fit", the reacting molecules are brought together at the
appropriate bonding sites. The enzyme, therefore, “lowers” the activation energy of the chemical reaction.
In a reaction catalyzed by an enzyme, the reacting molecules are called the substrate. The substrate
molecules combine with the active site of the enzyme forming a temporary complex called the enzymesubstrate complex. As the chemical reaction takes place and the products are formed, the enzyme is
released, unchanged from its original structure. Since the enzyme is not consumed or changed by the
chemical reaction, it can be used over and over to catalyze additional substrate molecules.
In this laboratory you will observe the activity of the enzyme, catechol oxidase, an enzyme that catalyzes the
conversion of the chemical catechol to a brown pigmented substance, benzoquinone. Catechol oxidase is
found in the cells of many organisms. You have probably observed this reaction a number of times when you
have cut potatoes or many fruits and left them out on the counter. They turn a rusty or brownish color. The
potato is a good source for this enzyme.
CLEAR
BROWN
Since catechol is colorless and the product, benzoquinone, is a rusty-brown color, the chemical reaction is
easy to detect. In addition, the intensity of the pigment produced is a reflection of the amount of catechol that is
converted. This, in turn, can tell you how effectively the catalyst, catechol oxidase, has worked under a set of
experimental conditions.
Form Fits Function
Some enzymes can only work when they have associate molecules, called coenzymes or cofactors present.
Many of our vitamins function as coenzymes, and some of our minerals, notably zinc, copper and iron, function
as cofactors. Heavy metals may interfere with enzyme function. Mercury, for example, inhibits the function of
many enzymes, partly by blocking the attachment of the needed cofactor. Substances that interfere with
coenzymes or cofactors will also inhibit the enzyme.
Copper is a cofactor for catechol oxidase.
This exercise will investigate the inhibition of enzyme activity by specific chemicals called inhibitors.
The specific inhibitor used will be phenylthiourea (PTU). To be active, catechol oxidase requires
copper as a cofactor. PTU is known to combine with the copper in catechol oxidase and inhibit its
enzymatic activity.
In this experiment we will examine the effect of shape on Enzyme function. We will measure the production of
Benzoquinone in the presence and absence of the Enzyme, in the presence of the Enzyme pre-incubated with
PTU, and the Enzyme pre-incubated with lemon juice.
Lemon Juice
In this part of the lab we will test the effect of lemon juice (different pH solutions) on the rate of
production of Benzoquinone.
Lemon Juice is commonly used to preserve fruit. This is because Lemon Juice contains a 5-6% Citric
acid solution which will denature the catechol oxidase, thus slowing the oxidation of Catechol to
Benzoquinone.
Table 1. Experimental Setup
Table 1 summarizes the experimental setup including the amounts of each solution for the Enzyme lab.
Experimental Setup
Cuvette
Condition
Catechol
Water
Enzyme
1
Normal Enzyme
4ml
0ml
1ml (full Strength)
2
Dilute Enzyme
4ml
0ml
1ml (1/10th strength)
3
no cofactor
Lemon Juice
(Diluted)
Lemon Juice
(Concentrated
Control (no Enzyme)
4ml
0ml
4ml
0ml
4ml
0ml
4ml
1ml
1ml (Pre-incubated in PTU)
1ml (Pre-incubated in diluted Lemon
Juice)
1ml (Pre-incubated with concentrated
lemon juice)
0ml
4
5
6
Materials Needed For the Entire Class:
Colorimeter
Colorimeter cuvettes
Distilled water
Pipettes
Stock solutions of
1% Catechol
Enzyme Catechol Oxidase (potato extract)
Enzyme Pre-incubated in Phenylthiourea (PTU)
Enzyme Pre-incubated in concentrated Lemon Juice
Enzyme Pre-incubated in diluted lemon juice
Catechol, and phenylthiourea are poisons and catechol, in concentration, is a mutagen. Use caution
handling these chemicals. Avoid contact with these solutions. If a spill occurs, immediately wipe up the spill
with dry paper towels and then use disinfectant solution on the spill site. Dispose of all towels in a designated
contamination location. Be sure to notify your instructor of the spill and your clean-up procedures. Your
personal safety and the safety of other students is most important.
Make serial dilutions by taking 10 ml of lemon juice to a graduated cylinder and bring to 100ml. Then
take 10 ml of that solution and bring it to 10 ml etc.
Measuring Production of Benzoquinone
1. Put 4ml of Catechol into Cuvette
2. Wipe outside of cuvette and Carefully put cuvette into colorimeter.
3. Carefully add 1ml of Enzyme (or water) into Cuvette and shut the colorimeter.
SIMULTANEOUSLY start program on iPad.
4. Take colorimeter readings every 15 seconds.
5. Take readings for 6 minutes.
After 6 minutes carefully remove the cuvette, clean it thoroughly and repeat the above steps for each
of the conditions in the experiment.
Data analysis.
Graph your raw data
Calculate your maximum rate for each condition.
Hypothesis:
1. If there is catechol added to potato juice (which contains the enzyme Catechol Oxidase), then it will
speed up the process of oxidation of Catechol into Benzoquinone.
2. If there is no potato juice (which contains the enzyme Catechol Oxidase), then the catechol will take a
much longer time to oxidize into Benzoquinone.
3. If lemon juice concentrate or Phenylthiourea (PTU) is added to the potato juice, then it will denature the
enzyme and then the catechol will take a much longer time to oxidize into Benzoquinone.
4. Diluted enzyme and enzyme pre-incubated with dilute lemon juice will have an intermediate effect.