Enzyme + Enzyme – Enzyme - Enzyme +
Substrate Substrate complex Product Complex Product
Catalase:
Catalase behaves as a catalyst for the conversion of hydrogen peroxide into water and oxygen. Catalase is an example of a particularly efficient enzyme. Catalase has one of the highest turnover numbers for all known enzymes (40,000,000 molecules/second). This high rate shows an importance for the enzymes capability for detoxifying hydrogen peroxide and preventing the formation of carbon dioxide bubbles in the blood
Catalase is composed of four subunits. Each subunit contains a heme group. This heme group is responsible for carrying out catalase's activity. Catalase functions to break down hydrogen peroxide (H2O2) into water and oxygen:
2H2O2 2H2O + O2
This reaction is performed by oxidation, the loss of electrons, and reduction, which is the gain of electrons. Each of the sub-units in Catalase uses the energy from electrons to decompose the Hydrogen Peroxide.
Catalase functions by the oxidation of Iron within its heme group. This happens by removing an electron from 2 molecules of hydrogen peroxide (H2O2) to form 2 water molecules (H2O) and 1 oxygen molecule (O2)
In Catalase, heme functions as a prosthetic group. A prosthetic group is a tightly bound, specific non-polypeptide unit required for the biological function of some proteins.
There are factors, which will control/affect the rate at which an enzyme-controlled experiment will take place. These include:
The Enzyme/Substrate concentration:
Providing conditions such as pH and temperature are at the normal for that specific enzyme, and there is an excess amount of substrate, then the arte of reaction will be directly proportional to the enzyme concentration. Increasing the amount of enzyme will increase the rate of reaction. If the amount of enzyme stays the same, the rate of reaction will increase with the increase in substrate concentration up to a point. When the enzymes active sites are all working as quickly as they can, adding more substrate would bring about no further increase in the rate of reaction.
PH:
Most enzymes have an optimum pH at which the rate of reaction is fastest. As we know the three-dimensional shape of an enzyme is vital for it to function properly. The most abundant chemical bonds found in the enzyme are the hydrogen bonds.
Small changes in the pH can affect the rate of reaction without denaturing the enzyme. At the extremes of its pH range, an enzyme can become unstable and denatured. Acidity and alkalinity can affect the active site of an enzyme. Free hydrogen or hydroxyl ions can affect the charges on the amino acid side chains of the enzymes active site. This will also affect the hydrogen bonds. If the bonds break due to a change in charge the three-dimensional shape will be lost, thus changing the shape of the active site. The substrate will no longer fit into the active site and will not be able to for an enzyme-substrate complex. Meaning that the enzyme looses its activity and the rate of reaction falls.
Low pH Correct pH High pH
Temperature:
Heating increases the rate of reaction in most chemical reactions. Heating a substance gives it greater kinetic energy, thus making the substance move around more. This means that there is a greater chance of molecules colliding, increasing the rate of reaction.
Increasing the temperature of an enzyme-controlled reaction will increase the rate o9f reaction but only up to a point. For most enzymes in the human body the rate will increase up to 40oc, a little higher than body temperature, known as the optimum temperature. This rise will bring about a corresponding rise in the rate of reaction. The optimum temperature is specific to the enzyme and has a lot to do where the enzyme is found.
Any increase in temperature will increase the energy of a single atom. This means that in an enzyme, the bonds between the polypeptide chain, would be also be affected. The rise would mean the bonds would begin to vibrate. Eventually the vibrating would get so fast, the bonds would break this will start to happen as the temperature increases past the optimum. The bonds that hold the precise three-dimensional shape will be broken, and so the precise shape will also be lost. This means that the active site will have changed, and the substrate can no longer bond to the enzyme. We say that the enzyme has become denatured.
If the enzyme becomes denatured, it will no longer work, even if you cool it. If you cool the enzyme below the optimum temperature, the enzyme becomes inactive. The enzyme will work again once heated.
In our experiment we are going to be using the enzyme Catalase, found naturally in potato. In our experiment we are going to be observing what happens when you vary the temperature at which the enzyme is working at.
Method:
Variables – In this investigation, the variables that affect the activity of the enzyme, Catalase, were considered and controlled so that they would not disrupt the success of the experiment.
i) Temperature – As temperature increases, molecules move faster (kinetic theory). In an enzyme catalysed reaction, such as the decomposition of hydrogen peroxide, this increases the rate at which the enzyme and substrate molecules meet and therefore the rate at which the products are formed. As the temperature continues to rise, however, the hydrogen and ionic bonds, which hold the enzyme molecules in shape, are broken. If the molecular structure is disrupted, the enzyme ceases to function as the active site no longer accommodates the substrate. The enzyme is denatured.
To control this variable, the temperature was maintained at a fairly constant level that allowed the enzyme to work effectively (room temperature, approximately 23ºC). This was achieved by using a test tube rack and tongs to handle the apparatus so that the heat from my hands did not affect the Catalase.
ii) pH – Any change in pH affects the ionic and hydrogen bonding in an enzyme and so alters it shape. Each enzyme has an optimum pH at which its active site best fits the substrate. Variation either side of pH results in denaturation of the enzyme and a slower rate of reaction.
In this experiment, the pH was kept constant using a pH 7 buffer, selected to maintain a pH level suited to the enzyme by being equal to the natural environment of the enzyme (potato tissue).
iii) Substrate Concentration – When there is an excess of enzyme molecules, an increase in the substrate concentration, produces a corresponding increase in the rate of reaction. If there are sufficient substrate molecules to occupy all of the enzymes´ active sites, the rate of reaction is unaffected by further increases in substrate concentration as the enzymes are unable to break down the greater quantity of substrate.
To control the substrate concentration, identical quantities of the substrate were used for each reading. To ensure that this was measured precisely, 5ml syringes were used to accurately gauge to exact quantities.
iv) Inhibition – Inhibitors compete with the substrate for the active sites of the enzyme (competitive inhibitors) or attach themselves to the enzyme, altering the shape of the active site so that the substrate is unable to occupy it and the enzyme cannot function (non-competitive inhibitors). Inhibitors therefore slow the rate of reaction. They should not have affected this investigation, however, as none were added.
v) Enzyme cofactors – cofactors are none protein substances which influence the functioning of enzymes. They include activators that are essential for the activation of some enzymes. Coenzymes also influence the functioning of enzymes although are not bonded to the enzyme.
Unless enzyme cofactors were present in the potato tissue containing the Catalase, they were not included in this investigation and therefore would not have affected the rate of reaction and the results of this experiment.
vi) Enzyme Concentration – Provided there is an excess substrate, an increase in enzyme concentration will lead to a corresponding increase in rate of reaction. Where the substrate is in short supply (i.e. it is limiting) an increase in enzyme concentration has no effect.
I varied the enzyme concentration by altering the number of equal sized discs of potato that contain the Catalase, in the reaction. The greater the number of discs, the greater the enzyme concentration.
Apparatus –