Investigation into the effect on the rate of reaction of the breakdown of hydrogen peroxide caused by changing enzyme concentration.

*The bonding has distorts the reactant, so the bonds to be broken are weaker.

*Hydrophilic amino acids create a water free zone for reactions with non-polar molecules

*Acid and base amino acids make the transfer of electrons easier.

The first step in any enzyme-catalysed reaction is the formation of an enzyme-substrate complex. The substrate attaches to an area on the enzyme known as the active site. The chemical reaction takes place with the formation of the products. The enzyme is then free to react again with any available substrate.

Enzymes are also specific.  One enzyme is designed to work with one substrate, to either break it down, or build it up. Emil Fischer came up with the lock and key theory in 1894. For a key (substrate) to fit and work it must be provided with the right shaped lock (enzyme). The substrate must have the right 3D shape/ conformation to bind to the active site.

E        +                S                ES                E                +        P

A more recent idea is the induced fit hypothesis. A temporary change to the active site of the enzyme allows the substrate to fit into the enzyme. The enzyme then wraps around the substrate, possibly causing strain on bonds to be broken.

E        +                S                ES                E                +        P

Factors affecting Enzyme Action

pH

Most enzymes have an optimum pH close to 7, this is the normal pH found within cells. Enzymes that work outside of cells, extracellular enzymes, can have very different pH requirements. e.g. Pepsin, This enzyme is found in the stomach and works best in highly acidic conditions (pH 1-2) Changing the pH can effect an enzyme in one of two ways;
1. If the active site takes the form of charged ions, some values of pH are inhibitory, because they cause changes to the charge in the site, the enzyme can no longer interact with the substrate and so it can no longer function.
2. The enzyme molecule may change shape and become denatured, particularly at high pH values.

Temperature

Initially, enzyme action increases as temperature increases. This is due to an increase in kinetic energy of the particles in solution. The increase in kinetic energy leads to more collisions and therefore more enzyme-substrate complexes form. However, because enzymes are organic they have an optimum temperature, which corresponds to a maximum rate of reaction. Above this temperature the reaction cannot involve the enzyme because denaturation has occurred and the enzymes three-dimensional shape has been lost. Most enzymes have an optimum temperature between 40-50°C.

Many inorganic reactions would continue to show a rise in activity beyond this point because they do not involve enzymes. Some bacteria can survive in hot springs above 85°C and fish in the Antarctic have enzymes that can function efficiently at temperatures of -2 C

Substrate concentration

The rate of reaction for most enzyme-controlled reactions varies with the available substrate. Increasing the amount of substrate can give a corresponding increase in the rate of reaction, as the amount of substrate becomes excessive the rate becomes more dependant on the amount of enzyme available.

This is because at low substrate concentrations some of the active sites of the enzymes will be unoccupied. At high levels all of the active sites will be occupied and further increases in substrate concentration have no effect on the rate.

At that point, an increase in rate can only be achieved by increasing the amount of enzyme available.

Enzyme Concentration

Normally enzymes need only be present in minute amounts to speed up a reaction, however, if there are no other factors limiting the reaction, then increased enzyme concentration means a higher rate of reaction. Because more enzymes available means more active sites for collisions, however, if the reaction runs out of substrate the enzymes cannot speed anything up and there will be plenty of active sites left empty.

Enzyme Inhibitors 

Inhibition is when the action of an enzyme is slowed down by another substance. There are two types of inhibitors, Competitive and Non-Competitive.

Competitive inhibitors 

These are chemicals that are usually closely related to the true substrate. They combine with the active site of the enzyme but cannot react. When inhibitor and substrate are both present they 'compete' for the active site of the enzyme, this slows down the rate of the reaction.

ie:

Small amounts of inhibitor greatly effect the rate of reaction, but the effect of competitive inhibitors can be overcome by;
a) increasing the amount of substrate
b) reducing the concentration of inhibitor

Non-Competitive inhibitors 

These inhibitors do not combine with the active site of the enzyme and are unaffected by substrate concentrations. The most common include heavy metal ions, such as mercury (Hg2+) and silver (Ag+) ions. The inhibitors bind with the enzyme and indirectly produce a change in the shape of the active site, so the enzyme is unable to combine with the substrate correctly.

ie:

The region on the enzyme where the non-competitive inhibitor binds is known as the allosteric site, these sites are one of the most important ways of regulating enzyme activity.

Enzyme co-factors 

Many enzymes will only work if a non-protein substance, called a co-factor, is present within the active site. It often contributes to the chemical reaction; sometimes it acts as a source of energy, helping the reaction to occur. Metal ions can act as co-factors - e.g. Mg2+, Fe2+, Zn2+ and Cu2+. When functioning in the liver, Catalase picks up iron as a cofactor to aid it with the reaction.

 Alternatively the co-factor can be a small organic molecule. These are termed coenzymes and are often related to vitamins. Cofactors, like enzymes, can be used again and again. Some enzymes have the coenzyme covalently bonded to them in an almost permanent way, if this occurs the coenzyme is known as a prosthetic group. 

Regulation of enzyme activity 

Some enzymes can become damaging if they are active in the wrong place. For example pepsin is capable of destroying body cells, therefore stomach cells manufacture pepsin in an inactive called pepsinogen. The enzyme only becomes active when pepsinogen is exposed to a strong acid.

If a sequence of reactions must take place in a set order, the enzymes are often arranged closely together and in the correct order, to effectively 'pass-on' the product of their reaction. In fact most enzymes in cells are bound to membranes to control their activity. e.g. mitochondria and chloroplasts. Precise control is achieved by a process known as end product inhibition, or feedback inhibition. The final product acts as non-competitive inhibitors for one of the enzymes at the beginning of the pathway.

ie:

Investigation into the effect on the rate of reaction of the breakdown of hydrogen peroxide caused by changing enzyme concentration

The aim of this investigation is to determine what effect an increased or decreased concentration of the enzyme catalase has on the breakdown of hydrogen peroxide (H2O2)

 We are using potatoes as our source of catalase and changing the surface area exposed to the hydrogen peroxide.

The reaction can be shown in the equation:

                Catalase

2H2O2                                2H2O                +        O2

                        Catalase

Hydrogen Peroxide                        Water        +        Oxygen gas

Prediction

As the cut surface area of the potato increases, the amount of product given off by the reaction will also increase. After a while I believe the results will begin to remain the same unless I prevent the limiting factors limiting the experiment.

This is because there is a direct link between cut surface area of potato and amount of enzyme. This is because catalase is found in the cut surfaces of potato and so increasing the surface area of potato exposed to the same amount of hydrogen peroxide increases the concentration of the enzyme present. Increased enzyme concentration means a higher rate of reaction because more enzymes available means more active sites are available for collisions to occur.

A sketch graph of the results we can expect would look something like this:

If the limiting factors, such as volume of substrate, are allowed to affect the experiment, then the results will even out, giving a results graph that looks like this:

The volume of hydrogen peroxide is a limiting factor because when it begins to run out the active sites of the enzymes are left empty because there is nothing left to react with it. From that point on it would not matter how much enzyme was present because without the substrate to react nothing would happen.

Preliminary work

All independent variables except for enzyme concentration must be kept constant to ensure a fair experiment. If they are not kept constant we will no longer be investigating the effects of enzyme concentration on the reaction, but instead the effects of all the variables on the reaction. This would make the investigation impossible to analyse or draw any conclusions from, because one effect would be indistinguishable from another. The dependant variable that we are measuring is volume of oxygen produced.

Temperature – For the preliminary investigation, room temperature is constant enough.

Volume of substrate – for each test a volume of 30cm3 H2O2 will be added

Mass of potato – This will be kept constant at 6g

pH – No extra acid or alkali  will be added to any experiment.

Shape of potato – the potato will be cut into 6 cubes of equal mass (6g), then sliced up according to test number:

Test 1, = 1 cube mass 6g

Test 2, = 2 cubes mass 6/2g each

Test 3, = 4 cubes mass 6/4g each

Test 4, = 8 cubes mass 6/8g each

Test 5, = 16 cubes mass 6/16g each

Test 6, = 32 cubes mass 6/32g each

Time – Volume O2 collected will be recorded every minute until the reaction is over to see what is the best time to collect the results at.

Enzyme Concentration – this is the variable being changed

Enzyme Inhibitors – None present

Enzyme co-factors – None present

Regulation of enzyme activity – None present.

 Apparatus needed

Water bath,

Knives,

Potatoes,

Round bottomed flask,

H2O2,

Syringe,

Measuring cylinder (10cm3 and 50cm3),

Bowl & Water,

Tubing and Bungs,

Method

1. Set up apparatus as shown. Use the 10cm3 measuring cylinder, ensuring it is filled completely with water. Do not attach the bung to the round-bottomed flask at this point.

2. Peel the potato and cut the six pieces of mass 6g. Slice them into their predetermined number of pieces and record their surface areas.
3. Measure out 30cm3 of H2O2 using the 50cm3 measuring cylinder
4. Add the first slice of potato to the round-bottomed flask.
5. Secure the syringe to the bung and the bung to the top of the round-bottomed flask.
6. Add the H2O2 from the syringe to the round-bottomed flask whilst simultaneously starting the stop clock.
7. Record the volume of O2 given off every minute
8. Repeat the experiment, missing out step three, and substituting step 5 for step 10 on test 2, step 11 on test 3, step 12 on test 4, step 13 on test 5 and step 14 on test 6
9. Add the second slices of potato (2 individual pieces)
10. Add the third slices of potato (4)
11. Add the fourth slices of potato (8)
12. Add the fifth slices of potato (16)
13. Add the sixth slices of potato (32)

After the tests have been carried out for the six pieces of potato, repeat the entire experiment three times to ensure a good set of reliable results are provided.

Calculating Surface Area

The surface area of a square, or rectangle, is the width multiplied by the height:

The surface area of a cube is:

• the width multiplied by the height of face A, PLUS
• the width multiplied by the height of side B, PLUS
• the width multiplied by the height of side C.                         

Calculating Rate of Reaction

The rate of reaction is given to be:

The rate of reaction = Change in an observable property

                                        Change in time

The observable property would be, in this case, the volume of O2 given off.

The decisions made in this experiment, such as mass of potato; volume of substrate; shape of potato and temperature, were made based upon preliminary work I have carried out. I have decided that for ease of calculation the shape of potato should be cubic; the volume of substrate needs to be excessive so it does not hinder the experiment; and the temperature should be around 37°C to allow the enzyme its optimum conditions.

        I decided to record the amount of oxygen gas given off after three minutes because if less time is used then the results are too similar for the higher enzyme concentration readings. If more time is used then the lower enzyme concentration tests have finished reacting before the time is up.

        It is also better to use not only the same type of potato, but instead, the same potato for each batch of tests because the concentration of catalase is not constant within all potatoes. It is more likely to be constant within potatoes from one plant, and it is very likely to be constant in one potato.

        The measuring cylinder used to collect the gas has to be the 25cm3 for all the experiments, because the 10cm3 is not big enough.

Risk Assessment

Although the investigation is not overtly dangerous, it is important to take all necessary precautions to eliminate the risk of hazards.

H2O2. This is a highly toxic chemical, so a lab coat and goggles should be worn at all times to protect clothes and eyes. It should be handled carefully in order to minimise the risk it poses. If contact with skin occurs the H2O2 should be washed off immediately because it is corrosive.

Knives. These should be handled carefully at all times, in order to lessen the chance of injury whilst cutting and peeling the potatoes.

Water bath. This is an electrical hazard; take care when switching it on.

Glass equipment. All care should be taken in order to diminish the possibility of breakages.

Revised Experiment

Independent variables

Temperature – the round-bottomed flask containing the potato (and H2O2 when it is added) will be in a water bath at a constant temperature of 37°C (mimicking body temp)

Volume of substrate – for each test a volume of 30cm3 H2O2 will be added

Mass of potato – This will be kept constant at 6g

pH – No extra acid or alkali will be added to any experiment.

Shape of potato – the potato will be cut into 6 cubes of equal mass (6g), then sliced up according to test number:

Test 1, = 1 cube mass 6g

Test 2, = 2 cubes mass 6/2g each

Test 3, = 4 cubes mass 6/4g each

Test 4, = 8 cubes mass 6/8g each

Test 5, = 16 cubes mass 6/16g each

Test 6, = 32 cubes mass 6/32g each

Time – Volume O2 collected will be recorded every minute for 4 minutes

Enzyme Concentration – this is the variable being changed

Enzyme Inhibitors – None present

Enzyme co-factors – None present

Regulation of enzyme activity – None present

Apparatus needed

Water bath,

Knives,

Potatoes,

Round bottomed flask,

H2O2,

Syringe,

Measuring cylinder (25cm3 and 50cm3),

Bowl & Water,

Tubing and Bungs,

Method

1. Set up apparatus as shown. For the first experiment use the 25cm3 measuring cylinder, ensuring it is filled completely with water. Do not attach the bung to the round-bottomed flask at this point.

2. Set the water bath to 37°C
3. Peel the potato and cut the six pieces of mass 6g. Slice them into their predetermined number of pieces and record their surface areas.
4. Measure out 30cm3 of H2O2 using the 50cm3 measuring cylinder
5. Add the first slice of potato to the round-bottomed flask.
6. Secure the syringe to the bung and the bung to the top of the round-bottomed flask.
7. Add the H2O2 from the syringe to the round-bottomed flask whilst simultaneously starting the stop clock.
8. Record the volume of O2 given off after three minutes.
9. Repeat the experiment, missing out step three, and substituting step 5 for step 10 on test 2, step 11 on test 3, step 12 on test 4, step 13 on test 5 and step 14 on test 6
10. Add the second slices of potato (2 individual pieces)
11. Add the third slices of potato (4)
12. Add the fourth slices of potato (8)
13. Add the fifth slices of potato (16)
14. Add the sixth slices of potato (32)

After the tests have been carried out for the six pieces of potato, repeat the entire experiment three times to ensure a good set of reliable results are provided.

After the tests have been carried out for the six pieces of potato, repeat the entire experiment three times to ensure a good set of reliable results are provided.