A catalase molecule is a tetramer of four polypeptide chains, with each chain comprising of over 500 amino acids. There are four porphyrin- haem groups in the tetramer that are very much like the familiar haemoglobins, cytochromes, chlorophylls and nitrogen-fixing enzymes in legumes. The haem group is responsible for catalase’s enzyme activity. Catalase has one of the highest turnover rates for all enzymes: one molecule of catalase can convert 6 million molecules of hydrogen peroxide, a powerful and potentially harmful oxidising agent, to water and oxygen each minute;
2H2O2 2H2O + O2
(Catalase also uses hydrogen peroxide to oxidize toxins including phenols, formic acid, formaldehyde and alcohols.
H2O2 + RH2 to 2H2O + R)
Hypothesis
In this experiment, I hope to be able to demonstrate the effects of copper ions on the activity of the enzyme catalase.
Copper ions have an effect on the activity of catalase as they are heavy metals and act as competitive inhibitors in the reaction between catalase and its substrate, hydrogen peroxide. The volume of oxygen that evolves will measure the effect of the addition of different concentrations of copper ions on the rate of the reaction.
Prediction
I predict that the volume of oxygen produced, will be at a slower rate as the concentration of the copper sulphate is increased, as copper sulphate is a competitive inhibitor of catalase. The copper ions will hence, compete with the substrate for the active site on the enzyme catalase.
I predict that the graph would look like this without the presence of the inhibitor;
Background Information
Enzymes are biological catalysts that speed up the rate of the reaction that would otherwise be too slow to support life. They are very large and complex organic molecules that are synthesized by the cell to perform very specific functions.
There are 5 main factors that affect the activity of enzymes and every enzyme has an optimal range foe each of these factors. Enzyme activity and as a result the rate of the reaction, is decreased if the enzyme is exposed to conditions outside this range.
As temperature increases so does the rate of reaction. This is because by increasing the temperature, the molecules have more kinetic energy, therefore they collide more frequently, forming more enzyme-substrate complexes. However once the optimum pH is reached, the rate of reaction begins to decrease as above this value, the shape of the active site is altered. This is because the high temperatures cause hydrogen bonds holding the active site in shape to break. An alteration in the shape of the active site means that the enzyme and substrate are no longer complementary so fewer and fewer enzyme-substrate complexes are formed Temperatures above 40-50°C denature many enzymes..
pH is measured on a scale of 0-14. Values below 7 are acidic, values above 7 alkaline and a value around 7 is neutral. As the pH moves into the acidic range the enzyme tends to gain hydrogen ions from the solution. As the pH moves into the alkaline range the enzyme tends to lose hydrogen ions to the solution. In both cases the changes in pH cause the charge on the R-group of the active site to change, denaturing the enzyme.
If all other conditions are held constant, the rate of the reaction should increase with increasing concentrations of substrate. At very low values of substrate the reaction rate will increase very rapidly. At higher substrate concentrations the rate begins to level off as there will be fewer active sites for the substrate to bind with. Eventually the maximum rate for that reaction will be achieved and further increases in substrate concentration will have no effect.
Every enzyme has an optimum salt concentration in which it can catalyze reactions. Too high or too low a salt concentration will denature the enzyme.
A molecule that interacts with the enzyme, reducing the rate of enzyme-catalysed reactions, as it has a similar shape to the substrate, is known as an inhibitor.
Competitive inhibition occurs when the inhibitor has a similar structure as the substrate, and competes for the active site on the enzyme. The degree to which they reduce the rate of reaction depends on the relative concentration of inhibitor and substrate. For example, if there is a lot of substrate and only a small amount of inhibitor, it is more likely the substrate will bind to the active site than the inhibitor. However, if there is more inhibitor than substrate then the rate of reaction could be significantly reduced. Cyanide is a competitive inhibitor because it binds to the active site in the catalase molecule.
A non-competitive inhibitor, binds to the enzyme somewhere other than the active site, often known as the ‘allosteric’ site. This causes the enzyme to change shape and it is left permanently denatured. Unlike competitive inhibition, the relative concentrations of inhibitor and substrate do not affect the degree of inhibition as the inhibitor will bind to the enzyme even if there are large amounts of substrate present.
picture
Variables – a variable is a parameter that can change
DEPENDENT VARIABLE
This is the variable that will be measures. It is known as ‘dependent’ as its value depends on the independent variable.
In this investigation, the dependent variable will be the volume of oxygen gas produced. This volume produced will depend on the rate of activity of the catalase’s ability, to covert the hydrogen peroxide to water and oxygen. The volume of gas produced will be measured using a gas syringe. The volume of gas produced will be measured every 10 seconds for 1 and a half minutes. To ensure accurate results, the volume of the gas will be measured to 2d.p. To ensure reliable results will be obtained, each concentration will be repeated three times.
This value depends on the concentration of the copper ions.
INDEPENDENT VARIABLE
This is the variable that will change and effects the volume of oxygen that is produced. This will be the concentration of copper ions used. Adding different volumes of distilled water to the copper ion solution will make different concentrations. The concentrations to be used are as followed;
- 0.001M
- 0.01M
- 0.1M
- 0.5M
- 1M (M = molarity)
Every solution will be of a certain volume (this will be specified after the pilot study has been carried out). The volume of solution will be measured accurately using a 10cm3 measuring cylinder and a pipette.
CONTOLLED VARIABLE
The volume of copper ions will be kept the same, as the aim of the experiment is to see how concentration affects the rate at which catalyst catalyses hydrogen peroxide to water and hydrogen. The temperature will be kept the same (ROOM TEMPERATURE) as if this is not controlled, it will affect the rate at which the enzyme works and will produce unreliable results, as a result the solution will be kept in a thermo regulated water bath for 5 minutes and all of the procedures, will be carried out in the same room on the same day. The amount of substrate, hydrogen peroxide, will be kept the same at 10cm³, as by altering the rate of reaction will change, as concentration of substrate affects enzyme activity. The pH also affects enzyme activity, so it must be the same (ph7) for all solutions. This can be controlled by buffering the solutions and checking their pH to make sure they are all the same.
OUTLINE METHOD
APPARATUS
materials
- Glass syringe ( cm³ ), this will be used to measure the volume of oxygen produced. It allows the volume to be read accurately to 1d.p. Collecting the volume of gas this way is more accurate and reliable than counting the number of bubbles produced in a boiling tube, which would be the alternate method.
- Conical flask, this will be used to put the hydrogen peroxide, copper(II) sulphate and celery solution into, as it provides a large for the substrate and enzyme to work in.