An investigation into the effect of substrate concentration on the activity of the enzyme catalase in potato tissue.

AS Biology Coursework Experiment.

An investigation into the effect of substrate concentration on the activity of the enzyme catalase in potato tissue.

Introduction.

Enzymes are globular protein molecules known commonly as Biological Catalysts. The definition of a catalyst is a substance that can be used to speed up the rate of a chemical reaction while remaining unaffected and unchanged after the reaction. The enzyme’s ability to act relies strongly upon the specific shape of the protein molecule, which is coiled into a precise three-dimensional shape. This specific shape is the cleft or depression, which other molecules (substrates) fit into perfectly; this part of the enzyme, is called the active site. The tertiary folding of the polypeptides causes the intricate specific shape of the active site. The substrates are held in the active site by forming temporary bonds with the hydrophilic R groups of the enzyme’s amino acids.

Here we have a diagram showing the ‘lock and key’ theory of enzyme action.

The substrate (key) is shown fitting perfectly into the active site (lock). The substrate binds to the enzyme and forms an enzyme-substrate complex. The reaction then takes place immediately and a substrate is either broken down into two or more products (catabolic reaction) or two or more substrates are bonded to make one product (anabolic reaction). The ‘induced fit’ theory states that initially the active site is no the perfect shape of the substrate but as it approaches, the shape of the active site changes to make the perfect fit.

Below is a sketched graph showing how enzymes reduce the amount of activation energy required for a reaction to occur; and hence speeding up the reaction.

This explains why enzymes are so vital in the human body. Some of our bodies reactions take place uncatalyzed at temperatures over 40*C which would damage our molecules unless we had enzymes to lower the heat energy required.

Variables: Factors that affect reaction rates.

There are five main factors affecting rates of reaction. They are:

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Enzyme concentration: if there are more substrate molecules then there are enzyme molecules, the available number of active sites becomes a limiting factor. The concentration factor reaches its optimum rate of reaction when all active sites are being used.

Temperature: when heat energy is added to the enzyme and substrate, random movement is increased. This means that when a lot of energy is transferred, there is an increase in molecule collision. When there are more collisions, there is more chance of the substrates finding their allocated active sites. This only works up to a certain ...

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Enzyme concentration: if there are more substrate molecules then there are enzyme molecules, the available number of active sites becomes a limiting factor. The concentration factor reaches its optimum rate of reaction when all active sites are being used.

Temperature: when heat energy is added to the enzyme and substrate, random movement is increased. This means that when a lot of energy is transferred, there is an increase in molecule collision. When there are more collisions, there is more chance of the substrates finding their allocated active sites. This only works up to a certain temperature, at which point the enzymes are denatured, this temperature is normally after 30 and 40*C which is where most enzymes are at their optimum.

Substrate concentration: when an excess amount of substrate is added to a constant amount of enzyme, the enzyme is soon working as hard as it can and each molecules active site is being used, this is known as Vmax. From this point, it is almost as if the substrate molecules are literally queuing up and waiting to fit into their allocated active site.

Levels of pH: The pH of the reaction does affect the secondary and tertiary structures of the enzymes. If this happens and the active site changes shape, it’s action will be affected. The majority of enzymes have optimum pH levels around neutral or are mildly alkali. Changes in pH, especially if acidic conditions occur, mean that the enzyme will not bind with the substrate, but neutralisation of the acid will generally make the active site normal again.

Surface area of substance containing enzyme: An increase in surface area means that there is a larger area and therefore larger number of exposed enzyme molecules to act. This increase in collisions causes an increase in rate of reaction and is therefore a factor that can limit rate of reaction and can also optimise reaction rates.

Planning and Prediction:

After studying the nature and behaviour of enzymes, I have decided to carry out my experiment varying the substrate concentration. In my preliminary experimentation I used a liver solution which contained the enzyme. Unfortunately, the liver contained a concentration of catalase which was too strong and meant that each experiment ended in a small explosion (so a set of results seemed hard to obtain). In this experiment I am going to use the enzyme catalase from potato tissue. So I am going to maintain a constant amount of the potato tissue (5cm ) for each trial. The enzyme catalase is used in living organisms to convert the toxic product hydrogen peroxide into water and oxygen. As the substrate, hydrogen peroxide, is reacted with the enzyme catalase, I am going to collect and measure the volume of Oxygen gas given off. I am going to use the water displacement method to collect the gas. I am going to make the oxygen gas displace some water in a measuring cylinder. The experiments were carried at a standard room temperature, and volumes, concentrations and quantities were measured to exactly the same values for each trial. Here is a predicted formula for the reaction:

2H202 catalase 2H20 + 02

I predict that an increase in the substrate (Hydrogen Peroxide 2vol) concentration will result in an increase in reaction rate. I also feel that after a certain point, the rate of reaction will decrease due to an excess of substrate and therefore a queue for reaction will occur. Catalase is able to speed up the decomposition of Hydrogen peroxide because the shape of its active site matches the shape of the Hydrogen peroxide molecule. This type of reaction where a molecule is broken down into smaller pieces is called an Anabolic Reaction.

Diagram of Apparatus

Method:

First of all I set up the apparatus as in the diagram. Using a cork borer, I cut five equally sized cylinders of potato tissue. I then placed each of these cylinders into their own test tubes. Attached to the bung of the tube was a delivery tube, which we could insert a syringe into. From the bung, there was another delivery tube that was long enough to reach another test tube. I placed the bung into a test tube with a cylinder in it, the delivery tube from the bung went to the measuring cylinder, which was filled with water and submerged in the water bath. Once this was all connected and set up, I filled the 10ml syringe with 2-vol. hydrogen peroxide. I then proceeded to inject the peroxide into the test tube with the potato. As soon as I had injected it, I started a stop- watch and let the reaction occur for three minutes. I then repeated the same process using the other potato cylinders and varying (reducing) the concentration of the hydrogen peroxide by diluting it with water.

To further my investigations of enzymes, I repeated my whole experiment but this time, more pieces of equally sized potato were cut, and I mashed the potato to a paste in order to maximise surface area. I then obtained all my results from the experiments and tabulated them.

From my table of results, I made three graphs. The first graph shows the volumes of Oxygen gas produced with different concentrations of the hydrogen peroxide with the solid potato cylinders. From this graph, we can see that there is an almost directly proportional increase in gas produced as the concentration increases.

In the second graph, I plotted the increase in volume of gas produced with different concentrations of hydrogen peroxide. This graph shows the results of my experiment using the mashed potato. Here we can see that there is instantly an increase in gas produced. This graph too shows the same pattern as the first graph, but because of the increased surface area, there is a larger variance in gas produced. This is evident from the larger spaces between the results.

In order to further my experiments and knowledge, I plotted a third graph, which has the two sets of results on the same graph. This enabled me to see the differences of using a larger surface area. This was another investigation in my experiment. I saw the same pattern in both graphs except for the presence of a clearer difference between the points, as two factors were used at the same time. This meant that the yield of gas produced was maximised.

The three following pages show my graphs.

From my results, it is possible to conclude that an increase in substrate concentration is (almost) at a constant with an increase in the rate of reaction. It is also possible to conclude that an increase in surface area, increases the rate of reaction as well.

Evaluation.

In my two sets of results, there are clearly a fair amount of anomalies due to school laboratory conditions, which differ to a more accurate set of results which would be obtained in a more expensive and extended science laboratory. Errors in accuracy are expected but if a line of best fit is imagined in my sets of results, it is clear that my results fall off the line only very slightly at a few points. These errors would be due to faulty/ inaccurate measurements of substrate and/ or catalase, differing temperatures, and surface areas not exactly the same. It is therefore possible to say that any imbalances in any of the factors affecting rate of reaction would have given me an inaccurate set of results. Higher levels of accuracy would have been maintained using thermometers to keep a constant temperature, pieces of potato exactly the same, measurements of volumes of liquid taken at higher accuracy’s, and time periods counted to an even more accurate level. As using catalase founded in potatoes, the desired amount was hard to measure, however, measuring the amount of potato wasn´t difficult although that piece could contain different amounts of catalase compared to another piece.

The theoretical maximum rate of reaction is when all the sites are being used but in reality this theoretical maximum is never reached due to the fact that not all active sites are being used at the same time. The substrate molecules need time to join onto the enzyme and to leave it so the maximum rate achieved is always slightly below the theoretical maximum. The time taken to fit into and leave the active site is the limiting factor in the rate of reaction.

Given the laboratory conditions, my results (although not perfect) give a clear indication of patterns and show clearly the effects of increasing substrate concentrations (along with increases in surface area).