Measuring enzyme-controlled reactions

Factors affecting enzyme-controlled reaction rates

Different factors can influence the rate of reaction, and these include:

• Temperature: with increasing temperature, the enzyme activity increases due to increased collisions between the molecules. However, too high of a temperature can cause an enzyme to denature.

• pH: different enzymes have different optimum pH levels that they can work best in. Changes in pH outside the optimum range can denature an enzyme.

• The concentration of the substrate: more substrate will equal more collisions with the enzyme; however, the rate does not continue to increase exponentially unless there is an unlimited number of enzymes. When all enzyme active sites are occupied, no more substrates can bind.

• The concentration of the enzyme: increasing the concentration of the enzyme will increase the rate of reaction up to a certain point unless there is an unlimited supply of substrate. When all substrate has been reacted with, the enzyme has nothing else to bind to.

• Presence of inhibitors: inhibitors can either inhibit the enzyme's active site (substrate cannot bind) or bind to the allosteric site (changes the active site).

• Enzyme activators: specific molecules that bind to enzymes to increase their activity.

Effects of substrate concentration

Investigating enzyme-controlled reactions - required practicals

Let's have a look at some examples of practicals you may encounter.

The effect of temperature on the rate of reaction

In this practical, the rate of reaction of an enzyme-controlled reaction will be investigated using a powdered milk suspension. The independent variable on this occasion will be temperature.

Material list

For this practical, you will need:

• Powdered milk suspension

• Trypsin solution (0.5 %)

• Distilled water

• Hydrochloric acid (HCl) (0.1 M)

You will use a variety of conical flasks, tubes and other equipment as needed. Your teacher will provide these.

Trypsin is an enzyme that aids with digestion. For this practical, trypsin will be the catalyst.

Method

First, we need to create our control and test solutions.

Making up control samples

Make two control samples using the steps below. These control samples will be used to compare to your test samples.

• Add 5 cm³ of milk suspension to two tubes.
• Add 5 cm³ of distilled water to one tube to represent no enzyme activity
• Add 5 cm³ of HCl to the other tube to hydrolyse (break down of water) the sample. This represents the presence of enzyme activity.

Making up test samples and measuring the rate of reaction

The goal is to measure the reaction rate; we will do this by timing how long it will take for the enzyme to affect the reaction at different temperatures. When all protein in the milk has reacted, the solution will become colourless.

1. Add 5 cm³ of milk into three test tubes and place all into a water bath at 10ºc for 5 minutes.
2. Add 5 cm³ of trypsin to each and start the timer.
3. Time how long it takes for the solution to become colourless.
4. Repeat steps 1 to 3 at different temperatures, such as 20ºc, 30ºc and so on.

Calculating the rate of reaction

To help with data-keeping, we suggest you use the following table arrangement:

You will have three repeats for each temperature (you can have more if you prepare more test tubes). By adding and dividing the time by the number of tubes, you will calculate the mean time taken for the sample to hydrolyse.

Now that you have timed all the reactions, you can calculate the rate of reaction using the following equation:

Rate of reaction = 1 / mean time

When you calculate rates of reactions for each temperature, you can plot them on the graph to visualise the reaction rates.

The effects of pH on the rate of reaction

Enzymes require a specific pH to work best. Enzymes have a narrow range, and the optimum activity will be at the highest rate. This practical will investigate the optimum pH for the amylase enzyme.

Amylase is a catalyst for the hydrolysis of starch (amylose) to maltose. It is mostly present in plant tissue, which includes seeds. Starch will be broken down during seed germination to release glucose, a food source for the growing seed. Amylase works best at a neutral pH of 7 and a temperature of 37 ° C.

Material list

• Amylase enzyme solution
• Starch solution
• pH solution
• Iodine solution

Method

1. Set up the bunsen burner with the tripod over it - this is where you will place your beaker with the solution.
2. Place the beaker containing water on the tripod above the bunsen burner and keep the temperature at 35 degrees (adjust the flame accordingly).
3. Add a couple of drops of iodine solution onto the spotting tile.
4. In the test tube - mix 2 cm³ of amylase, 2 cm³ of starch and 1 cm ³ of pH solution.
5. Place the test tube in the beaker with water.
6. Every 20 seconds pipette a few drops of solution on the spotting tile (containing iodine solution).
7. Do this until the iodine solution no longer turns black, and note the time.
8. You can repeat this with different pH solutions.

The effect of changing substrate concentration on rate of reaction

During cellular respiration, your mitochondria produce hydrogen peroxide (H₂0₂) as a by-product of oxygen combustion (substances react with oxygen to give off heat). Hydrogen peroxide is toxic to cells, so how does the body get rid of it to avoid harm? Several enzymes, including catalase, break down hydrogen peroxide. When it is broken down, it produces oxygen and water.

In this practical, you will investigate how different concentrations of hydrogen peroxide affect oxygen production.

Material list

• Hydrogen peroxide at a range of concentrations (example concentrations can include: 1M, 0.8M, 0.6M, 0.4M, 0.2M)
• Potato which has been cubed (this will act as the source of catalase)
• Large bowl
• Measuring cylinder

Method

1. Weigh the potato pieces and place them in the conical flask filled with 5cm³ hydrogen peroxide.
2. Fill a large bowl with water.
3. Fill the measuring cylinder and place it upside down inside the large bowl (make sure that no air bubbles are present).
4. The bung in the lid of the conical flask will have a tube extending out, which will go under the measuring cylinder.
5. Record the volume of oxygen in the measuring cylinder at regular time intervals (e.g. every 10 seconds).
6. Repeat with other hydrogen peroxide concentrations.

Increasing the concentration of hydrogen peroxide, i.e., the substrate will increase the reaction rate. However, this will not go on forever (unless an unlimited amount of enzyme is available), so when all enzyme active sites are occupied, no more substrate can be converted.

The effect of changing enzyme concentration on the initial rate of reaction

Another factor affecting the reaction rate is enzyme concentration. The more enzymes you have, the quicker the reaction rate until all active sites are occupied. In this practical, you will measure the absorbance using a colourimeter (measures light transmittance).

Material list

• Milk powder solution

• Trypsin solution (1%)

• Distilled water

Method

1. Dilute the stock trypsin solution to produce concentrations of 0.2, 0.4, 0.6 and 0.8 %.

2. In the control solution, add 2cm³ of stock trypsin (1%) and 2cm³ of distilled water. This solution will be used to set the absorbance to zero.

3. In your test solution, add 2cm³ of milk suspension and 2cm³ of stock trypsin (1%). Mix and place in the colourimeter to read the absorbance.

4. At regular 15 second intervals, record the absorbance reading. Do this for 5 minutes.

5. Now repeat this for the other trypsin concentrations.

You can use the rate of reaction formula in the previous section to calculate the reaction rate.

The initial reaction rate will be directly proportional to the concentration of the enzyme. If unlimited substrate was present as the enzyme concentration increases, this increase could continue forever!

When you plot the enzyme concentration against the reaction rate with your result, the points may not totally fall in a straight line. However, in the perfect world, this is how it will look (Figure 3).