An investigation to determine how the rate of an enzyme-catalysed reaction is affected by the availability of the active site of the enzyme during the reaction by using the enzyme catalase to decompose hydrogen peroxide.

Rishi Patel Planning for AS coursework on

“ An investigation to determine how the rate of an enzyme-catalysed reaction is affected by the availability of the active site of the enzyme during the reaction by using the enzyme catalase to decompose hydrogen peroxide”

Aim

The aim of this investigation is to determine how the rate of an enzyme-catalysed reaction is affected by the availability of the active site of the enzyme during the reaction. The effect on the rate of the reaction by the availability of the active sites on the enzyme catalase to decompose hydrogen peroxide will be studied.

Safety precautions

It is important to wear safety spectacles and plastic gloves at all times when carefully handling the hydrogen peroxide as it is extremely irritant to the skin and eyes. It is necessary to be careful when cutting tissues with the blade, which should be put away in its case when not in use. It is also necessary to follow the standard laboratory rules and take care not to touch the water bath once it has been heated up.

Scientific knowledge

A catalyst is a substance, which speeds up a chemical reaction but remains unchanged at the end of the reaction. All metabolic reactions in living cells are catalysed by enzymes. These are biological catalysts made of globular proteins, coiled into precise 3D shapes with the hydrophilic R groups on the outside of the molecule; therefore enzymes are soluble in aqueous solutions. The chemical/ chemicals, which an enzyme acts on, is called its “substrate”. The area where the substrate binds to the enzyme is called the “active site” of the enzyme. This is usually a cleft or depression and has a specific shape. This allows the substrate to fit perfectly and the combined structure is called the “enzyme-substrate complex”. The shape of the active site allows only one shape of molecule to fit exactly and hence the enzyme is said to be “specific” for this substrate. This is often referred to as the “lock and key” hypothesis. The active site is usually only between 3-12 amino acids of the large globular enzyme molecule.

Diagram To show the “lock and key” model of enzyme action.

The substrate fits exactly into the active site of the enzyme. The enzyme then catalyses the breakdown of the substrate into products, which then leave the active site of the enzyme.

The formation of the enzyme-substrate complex decreases the “activation energy” that the substrate needs to be converted to a product. The enzyme may catalyse a reaction in which the substrate molecule is split into two or more molecules or it may catalyse the joining together of two molecules. e.g. when making a dipeptide. Once the products have formed, they leave the enzyme’s active site and the enzyme is then free to combine with a new substrate molecule.

The rate of enzyme controlled reactions is always fastest at the beginning and is called the “initial rate of reaction”. This is because in the presence of excess substrate molecules, virtually every enzyme molecule will have a substrate molecule in its active site. The rate then depends on the number of enzyme molecules, the speed at which the enzyme converts the substrate to product, releases it and binds to another substrate molecule. As more and more substrate is converted into product, there are fewer and fewer substrate molecules to bind with the enzymes; the reaction slows down and finally stops.

In this investigation the concept of the “lock and key” model will be used to determine how the rate of the enzyme catalysed reaction is affected by the “availability” of the enzyme (i.e. how much the active sites (locks) are “exposed” to bind with the substrate molecules (keys))

It is best to study the effects on the “initial” rate of reaction because as the reaction progresses, the amount of substrate varies as it is converted to products at different rates.

Various factors affect the rate of enzyme catalysed reactions, such as:

Temperature: Between 0-40 C the rate of enzyme activity increases as more molecules have increased energy for successful collisions between enzyme and substrate molecules. As temperature increases above 40 C, the hydrogen bonds in the enzyme (protein) break and the enzyme looses its shape and activity- it is said to be “denatured”. This effect is irreversible. All enzymes have an “optimum temperature” at which the enzyme activity is at its maximum.
pH: Most enzymes work fastest at a pH of about 7 (but some enzymes like protease ...

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Various factors affect the rate of enzyme catalysed reactions, such as:

Temperature: Between 0-40 C the rate of enzyme activity increases as more molecules have increased energy for successful collisions between enzyme and substrate molecules. As temperature increases above 40 C, the hydrogen bonds in the enzyme (protein) break and the enzyme looses its shape and activity- it is said to be “denatured”. This effect is irreversible. All enzymes have an “optimum temperature” at which the enzyme activity is at its maximum.
pH: Most enzymes work fastest at a pH of about 7 (but some enzymes like protease work best in an acidic (low pH) environment). All enzymes have an “ optimum” pH at which the rate is fastest.
Concentration of substrate: As substrate concentration increases, the rate initially increases, but as all the active sites on the enzyme become “saturated” with the substrate the rate then depends upon how fast the products leave the active sites.
Concentration of the enzyme; In the presence of excess substrate the rate is directly proportional to enzyme concentration but as the amount of substrate decreases due to enzyme activity, the rate decreases.

The rates of enzyme catalysed reactions can be increased by factors of 10 to 10 compared to uncatalysed reactions. e.g. one molecule of catalase can catalyse the decomposition of about 600 thousand molecules per second of hydrogen peroxide at body temperature.

Catalase is an enzyme found in the tissues of most living things and catalyses the decomposition of hydrogen peroxide, a toxic by-product of some metabolic reactions into water and oxygen, which are both, harmless.

catalase

2H2O 2H2O + O2

Catalase is highly specific for the hydrogen peroxide molecule and can easily be used to investigate the rate of enzyme catalysed reactions by measuring the rate at which oxygen (the product) is released.

In this investigation I shall study the rate of decomposition by catalase of hydrogen peroxide by changing the surface area of tissue sample (hence changing the availability/exposure of the active site) and measuring the rate at which oxygen is released. There are many sources of catalase e.g. liver, potato.

Potato will be used, as it is easier to handle.

The factors that will be controlled will be:

Temperature: The experiment will be carried out at room temp (about 25 C) and all results taken on the same day in the same room to minimise errors due to temperature variations. (see later in “method”- the experiment will be conducted with all apparatus immersed in a “water bath” maintained at 35 C, the “optimum temperature” for catalase).
pH: All apparatus used will be washed out in distilled water initially (neutral pH of 7) to ensure there are no traces of chemicals left over from previous use of the apparatus to minimise errors.
The substrate; The same volume (40ml) of hydrogen peroxide of strength 20 vol will be used from the same stock bottle to keep the substrate concentration the same.
Concentration of the enzyme- The same dimensions of potato chips will be cut from the same potato and used, to ensure that the total amount of catalase is constant. The potato chips will have to be large enough to ensure there is enough catalase in them to react with the hydrogen peroxide.

However, I shall alter the surface area (the variable) of each potato chip by cutting each chip into pieces of pre-determined sizes (see later in preliminary test on how to improve the procedure). Hence, although the total amount of catalase in each chip remains constant, by altering the surface area I shall alter the availability/exposure of the active sites on the catalase. I shall study how this affects the rate of decomposition of the hydrogen peroxide into water and oxygen (which will be measured at pre-determined times-see preliminary on how to improve the procedure).

Hypothesis

As the surface area of the potato chips increases, more active sites of catalase will be exposed to the substrate, hydrogen peroxide. Catalase-hydrogen peroxide complexes will form at a faster rate and hence the product (water and oxygen) will be produced at a faster rate also. Once the product leaves the active sites, they will be available again for more substrate molecules to bind again and the catalysis continues until all the hydrogen peroxide has been converted into water and oxygen.

Prediction

I predict that the rate of reaction will be proportional to the “ availability” of the active sites on the catalase until all the hydrogen peroxide has been used up, as long as the temperature, pH, and total enzyme concentration remain the same.

A preliminary experiment was carried out so errors in the actual investigation can be minimised and further improvements to the procedure and sampling techniques can be made.

Preliminary experiment

For the preliminary experiment the apparatus was set up as shown in the diagram below:

A sample of potato tissue was taken using a cork borer and a 2cm wide chip, measured with vernier callipers, was cut with a sharp blade and placed in the conical flask.

40mls of 20vol hydrogen peroxide was added via the measuring funnel and the volume of oxygen released into the gas jar measured at 15seconds, 30secs and at 45secs, from when the hydrogen peroxide was added to the potato chip.

Another 2cm wide potato chip sample was taken from the same potato using the same cork borer but this time the chip was cut in half so that two 1.0cm wide pieces with the same diameters were obtained. The above procedure was repeated but both the pieces were added to the conical flask. Another 2cm wide potato chip sample was similarly cut into three pieces, each 0.66cm wide. The above procedure was repeated, but with the three pieces added to the conical flask. The results were noted as shown in the table below.

A table to show the volumes of oxygen obtained at various times when 40mls of 20vol hydrogen peroxide was added to different samples of potato chips

It can be seen from the results above that when the 2cm wide chip is cut into further pieces, the volume of oxygen measured at each time period increased. This suggests that as the surface area increases, the reaction occurs faster as expected, suggesting that more active sites on the enzyme are available, as the surface area increases.

For the actual investigation, some improvements will be made to the procedure:

The conical flask, measuring funnel, the 50ml measure used to measure the hydrogen peroxide, the rubber bung, the blade and tile used for cutting the potato will all be rinsed out with distilled water to ensure that the pH remains neutral and is not affected by any traces of chemicals left over from previous use of the equipment.

The volume of oxygen obtained will be measured at 15seconds, 30 secs, 45 secs and also at an extra time of 60secs from the addition of the hydrogen peroxide to obtain a larger range of results.

The surface area (and hence the “availability” of the active sites) of the potato chips will be varied at least five times and the procedure repeated twice (i.e. three results) for each surface area so a larger range of results can be obtained and the average volume of oxygen obtained can be calculated. 3cm instead of 2cm wide potato chips will be used, then each chip cut into further pieces to increase the surface area.

Larger size chips will be used to make sure there is enough total catalase to react with 40mls of 20vol. Hydrogen peroxide.

The width of the potato pieces will be as follows: (but all with the same diameter and circumference as the same cork borer will be used)

Sample no (1)- ONE 3cm wide piece

Sample no (2)- TWO 1.5cm wide pieces

Sample no (3)- THREE 1.0cm wide pieces

Sample no (4)- FOUR 0.75cm wide pieces

Sample no (5)- FIVE 0.6cm wide pieces

This sampling will be repeated twice and all samples will be obtained from the same potato.

A control will also be done in which everything remains the same but no potato sample will be present in the conical flask.

PLANNING FOR METHOD:

EQUIPMENT:

20vol Hydrogen Peroxide

All 40ml measures of hydrogen peroxide to be used in the experiment will be taken from the same stock bottle.

Distilled Water

This is used instead of tap water to rinse out equipment as necessary to avoid the introduction of other contaminants.

Large Potato

Stop Clock

To accurately measure the time from when the hydrogen peroxide is added to the potato sample.

Forceps

This will allow the potato to be handled without touching it as contaminants from fingers can affect the reaction between the hydrogen peroxide and the catalyse in the potato.

50ml Measuring cylinder

To measure the volume of hydrogen peroxide which is going to be added to the potato samples. This piece of equipment can measure to the nearest 1ml so the accuracy is ± 0.5ml.

Cork borer

To obtain potato tissue samples which are all of the same diameter and circumference.

Vernier Callipers

To accurately measure the width of each potato piece. This has an accuracy of ± 0.1mm.

Sharp Blade

To easily cut the potato pieces with minimal tissue damage.

White Tile

To place the potato sample on when cutting.

Measuring Funnel with valve

To hold the hydrogen peroxide before adding it to the potato sample.

Rubber Bung with two holes

Delivery Tube

Metal Clamp

A Beehive - to hold the delivery tube in place and allows the gas jar to be inverted on top of it.

Thermometer

To double check the temperature of the water in the water bath and this has an accuracy of ±0.5 C

Glass conical flask

To hold the potato samples

Electrical Water Bath

This has to be big enough to hold all the apparatus and is set at 35 C. Digital temperature sensors keep the water bath at the set temperature.

A 100ml Gas Jar

To collect the oxygen released and accurately measure its volume. This has an accuracy of ±0.5ml.

METHOD:

All safety precautions will be taken as stated under “safety precautions” of this planning.

The water bath will be filled with tap water, set at 35 C and switched on so the water warms up to 35 C. (Digital temperature sensors in the water bath will ensure this temperature is maintained.) A thermometer will also be placed in the water bath to double check that the 35 C temperature is maintained. The conical flask, rubber bung, blade, tile, forceps, measuring funnel, and the 50ml measuring cylinder will all be rinsed thoroughly with distilled water to remove any traces of other chemicals left over from previous use. A 100ml-gas jar will be completely filled up with tap water and then inverted on top of the beehive in the water bath.

40mls of 20vol hydrogen peroxide will be accurately measured using the 50ml measuring cylinder and placed in the water bath for 5-10 minutes so the hydrogen peroxide is also at about 35 C.

The apparatus will be set up as shown in the diagram for the “preliminary” experiment. The water bath will be filled with sufficient tap water to immerse the conical flask. A large potato will be peeled, washed in distilled water and a sample taken using the cork borer, and placed on the tile using forceps. A 3cm wide potato chip will be measured using vernier callipers and cut with the sharp blade. This sample (number 1) will be placed in the conical flask using forceps, replacing the rubber bung with the measuring funnel and delivery tube as soon as possible.

The 40ml of 20vol hydrogen peroxide will be placed in the measuring funnel, making sure the valve on the measuring funnel had been closed before adding the hydrogen peroxide. The valve on the measuring funnel will be opened to allow the hydrogen peroxide to be added to the potato sample and the stop clock started. The valve will be re-closed to stop any oxygen released from escaping through the valve.

The volume of oxygen collected in the gas jar will be measured at 15seconds, 30secs, 45secs, and at 60secs, using the stop clock, from the addition of the hydrogen peroxide and noted in the table below.

The conical flask, rubber bung and the measuring funnel will be washed out with distilled water after discarding the 3cm wide potato chip already used.

The procedure will be repeated twice to obtain three results and the average volume of oxygen collected worked out for each time and noted in the table.

Another 3cm wide potato chip will be cut as before using the same cork borer, blade and tile from the same potato but this time it will be cut again to obtain two pieces each 1.5cm wide with the same diameter and circumference. The above experiment will be repeated but with both these two 1.5cm wide pieces being placed in the conical flask together.

This will be regarded as sample number 2. This procedure will be repeated twice as before and the results noted.

The experiment will be repeated where:

Sample number 3 will consist of three, 1.0cm wide pieces.

Sample number 4 will consist of four, 0.75cm wide pieces.

Sample number 5 will consist of five, 0.60cm wide pieces.

The procedure will be repeated three times with each sample, the results noted and the averages calculated as before.

A control experiment will be done but no potato will be added to the conical flask.

From the results, graphs will be plotted of the average volume of oxygen collected/mls on the y-axis and time/second from when the hydrogen peroxide is added on the x-axis. for each sample number.

The following table will be used to note all results:

Secondary Sources

BIOLOGY 1: M.Jones et al Cambridge university press 2001

BIOCHEMISTRY: R.Fosbery Cambridge university press 2002

BIOLOGICAL SCIENCE 1: D.J.Taylor et al Cambridge university press 2001

PRE-EVALUATION

From the experiment there will be some inaccuracies:

The 50ml measuring cylinder used to measure the hydrogen peroxide will have a percentage error of

0.5ml X 100 = 1.25%

40ml

Vernier Callipers- used to measure the potato pieces will have a percentage error of

0.1mm X 100 = 0.33% if a 3cm width measurement is done

30mm

0.1mm X 100 = 0.66% if a 1.5cm measurement is done

15mm

Therefore the smaller the measurement the greater the percentage error.

Gas jar – used to measure the volume of oxygen collected will have a percentage error of

0.5ml X 100 = ?

Volume of oxygen collected in mls

The smaller the volume of oxygen collected the greater the percentage error.

Thermometer – used to double check that the temperature in the water bath remains at 35 C. This will have a percentage error of

0.5 x 100 = 1.43%

An investigation to determine how the rate of an enzyme-catalysed reaction is affected by the availability of the active site of the enzyme during the reaction by using the enzyme catalase to decompose hydrogen peroxide.

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