Investigating the Effect of Substrate Concentration on Catalase

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Naomi Laskar

Investigating the Effect of Substrate

Concentration on Catalase

Aim

To investigate the effect of substrate concentration the enzyme catalase using the decomposition reaction of hydrogen peroxide and measuring the volume of oxygen produced.

Hypothesis

Enzymes are globular protein molecules, which catalyse chemical reactions and therefore speed up the rate of the reaction and yet remain unchanged (Jones, Fosbery and Taylor, 2000). The enzyme’s structure, as it is three-dimensional, is determined by the sequence of amino acids. A few of these amino acids make up an active site. This is a cavity in which other molecule(s) can bind; this molecule is known as the substrate. The substrate is held in the active site by hydrogen bonds that temporarily form between the hydrophilic R-groups of the active site and on the substrate molecule. The structure that results is the enzyme-substrate complex.

The compounds on which the enzymes act upon are called substrates. The enzyme could either catalyse a reaction to breakdown into two or molecules or it could help to create bonds in the substrate molecules. The molecules that form from either of these reactions are called products. The activation energy is the initial energy needed for a reaction to occur. Enzymes lower the activation energy in order for reactions to happen more easily. Enzymes are used for reactions that take place very slowly or would normally occur at very high temperatures.

Each enzyme has a specific shape (the lock), which only a specific substrate (the key) can attach to, making enzymes specific to the reactions that they catalyse (Toole and Toole, 2004). This is the lock and key theory. However, the actual process is slightly more complicated than this and is known as the induced fit theory. The enzyme is slightly flexible and can therefore mould itself to fit perfectly around the substrate. As the shape is changed, the activation energy is lowered as a strain is put on the substrate molecule. The collision theory explains how reactant particles move towards one another and collide to break the existing bonds. It is concerned with the number of times the reactant particles hit each other and cause an induced fit.

The enzyme catalase catalyses the decomposition reaction of hydrogen peroxide:

2H2O2                 O2 + 2H2O

Catalase breaks the bonds in the molecules of hydrogen peroxide to release two molecules of water and oxygen. This oxygen will be measured in the following experiment by measuring the displacement of water. The more oxygen produced, the more water will get displaced. I predict that as the substrate (hydrogen peroxide) concentration increases, so will the amount of oxygen produced and therefore, the volume of water displaced will increase too. This is because substrate concentration affects enzyme-catalysed reactions. The enzyme amount stays fixed throughout the reaction as more and more substrate is added. When substrate concentration is low, the enzymes have only a few substrate molecules they need to breakdown. However as the concentration of hydrogen peroxide increases, the ratio of substrate molecules to enzymes becomes more equal and therefore the active sites work at their optimum speed and the rate of reaction reaches its maximum. After this point is reached, the rate of reaction levels off as the substrate will be in excess and the active site cannot work any faster.

This graph shows the rate of reaction with increasing substrate concentration. The substrate concentration is directly proportional to the initial rate of reaction. This is because when the substrate concentration doubles, so does the amount of oxygen that it decomposes into.

 

Preliminary Experiment/ Secondary Sources

The preliminary experiment was to investigate the effect of pH on the enzyme catalase.12cm3 of H2O2 and 10cm3 of pH 2, 7 or 9 was added to a conical flask. A delivery tube was placed from the bottom of a burette in a trough of water into the conical flask. 2cm3 catalase was then added, the bung was placed on the conical flask and the stopwatch was started.

(Preliminary Results Table, Discussion and Evaluation)

   

These results show that as the pH increased, the volume of oxygen produced also increased. Even though the optimum pH was pH9, I will not use pH9 for my actual experiment as it is too quick and therefore it makes it harder to read the volume of oxygen from the burette. As shown on the graph, pH2 was far too slow due to denaturalisation (Jones, Fosbery and Taylor, 2000). Hence, it would not be sensible to use pH2. The optimum pH for catalase is pH7 () and my preliminary results show that it will produce results similar to that of my predicted results (as demonstrated in the background knowledge) therefore I shall use pH7.

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The recording intervals of 15 seconds is quite a good length of time as it enabled enough oxygen to be produced for a clear graph to be plotted. The trials will last for 180s rather than 120s because some of the concentrations that will be used, for example 0.5% may be quite slow and so a better range of data can be collected if results are recorded for 180 seconds. To ensure there is a complete reaction, the conical flask will be swirled once, this will mix the reactant particles together thoroughly. The concentrations will range from 0 to ...

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