Zo Bradshaw Lower Science I Title: The effect of temperature on enzyme activity.

Date: 14th January, 2013 Aim: To determine the effect of temperature on the activity of catalase. Apparatus and Materials: Boiling tubes, measuring cylinder, thermometer, beaker, syringe, bunsen burner and tower, stopwatch, ruler, water baths at 35 oC and 70oC, ice, water, potato extract, hydrogen peroxide Introduction: Enzymes are protein molecules that can be defined as biological catalysts catalysts are substances that increase the rate of chemical reactions without being used up. This means that they make biochemical reactions happen faster than they would otherwise. Enzymes, by functioning as catalysts, serve to reduce the activation energy required for a chemical reaction to take place. The activation energy is the energy required for a chemical reaction to take place. Sometimes the essential reactions would not happen at all without the help of enzymes. Being catalysts also means that enzymes are not part of the final product. They make things happen. When the job is done, enzymes are ready to catalyse a new biochemical reaction. Enzymes are extremely efficient, this means that a tiny amount of catalyst brings about the change of a large amount of substrate. The presence of enzymes does not alter the nature or properties of the end product/products of the reaction. Enzymes are also highly specific, this means that they have one function and one function only. Only when the right enzyme finds the right substrate can biochemical reactions occur; an enzyme is so specific that it will generally catalyse only a single reaction. Every function or substrate in an organism has its own unique enzyme. Substrates are the substances that are to be changed during the reaction. The substrate which is to be transformed fits the enzyme accurately, enzymes usually catalyse reactions by providing a place for the substrate to fit, this place is known as the active site and it is this which has the specific shape. There are two hypotheses that explain how this works. The first one, suggested by Fischer in 1890 is known as the lock and key hypothesis. This theory suggests that the substrate is like a key whose shape is complementary to the enzyme which would be the lock, the substrate would then bind to the active site of the enzyme. The second theory, a modification to the lock and key model was

suggested by Koshland in 1959, this model was know as the induced fit hypothesis. This theory suggested that enzymes and their active sites were more flexible than how they were described in the lock and key theory, instead, it was thought that as substrate molecules fit into the active site to form a complex, the enzyme molecule changed shape slightly to move itself around so that the active site fitted precisely around the substrate. All known enzymes are proteins, specifically globular proteins and can occur in the body in very small amounts. Globular proteins have a tertiary structure and are maintained by the interactions of ionic, hydrogen and disulphide bonds as well as, hydrophobic interactions. Enzymes catalyze all processes in the body, enabling organisms to build up chemical substances such as other proteins, carbohydrates or fats that are necessary for life. In short, all enzymes are proteins, but not all proteins are enzymes. If a protein can catalyse a biochemical reaction, it is an enzyme. Being proteins, the structure and function of the enzyme is determined by the order of the amino acids. No two enzymes are alike. Each enzyme has its own unique sequence of amino acids, which is determined by the genes in the cells i.e. enzymes are coded for by DNA. The activity of enzymes is affected by various conditions such as pH, temperature, substrate concentration, enzyme concentration, inhibitors and incubation time. These conditions can result in the rate of reaction increasing or decreasing. All enzymes have an optimum pH. If the pH changes much from the optimum, the chemical nature of the amino acids can change. This may result in a change in the bonds and so the tertiary structure may break down. The active site will be disrupted and the enzyme will be denatured. As with the pH, enzymes work best at an optimum temperature. The rate of an enzyme catalysed reaction increases with temperature because an increase in temperature provides more kinetic energy to the molecules involved. The numbers of collisions between enzyme and substrate will increase so the rate will too. Above the optimum temperature, and the enzymes are denatured. Bonds holding the structure together will be broken and the active site loses its shape and will no longer work. For a given enzyme concentration, the rate of an enzyme reaction increases with increasing substrate concentration. At a low substrate concentration there are many active sites that are not occupied. This means that the reaction rate is low. When more substrate molecules are added, more enzyme-substrate complexes can be formed. As there are more active sites, and the rate of reaction increases.

Eventually, increasing the substrate concentration yet further will have no effect. The active sites will be saturated so no more enzyme-substrate complexes can be formed. This is why theoretical maximum rate, Vmax, is never quite obtained. When enzyme concentration is low there is great competition for the active sites and the rate of reaction is low. As the enzyme concentration increases, there are more active sites and the reaction can proceed at a faster rate. Eventually, increasing the enzyme concentration beyond a certain point has no effect because the substrate concentration becomes the limiting factor. Inhibitors are substances that slow down the rate of an enzymecontrolled reaction. Sometimes inhibition is a necessary way of making sure that the reaction does not proceed too fast, at other times, it is undesirable. There are two types of inhibitors, reversible and irreversible. With reversible inhibitors there are also two types, competitive and non-competitive. In competitive reversible inhibition the molecules have a similar structure to the actual substrate and so will bind temporarily with the active site preventing the true substance from entering. The rate of reaction will therefore be closer to the maximum when there is more 'real' substrate. Non-competitive reversible inhibitors are not necessarily anything like the substrate structurally. They bind with the enzyme, but not at the active site. This does not affect the ability of the substance to bind with the enzyme, but it makes it impossible for catalysis to occur. The reaction rate decreases with the increase of inhibitor concentration, no reactions occur when the inhibitor saturation has been reached. Unlike competitive inhibition an increase in substrate concentration does not affect the rate of reaction. Irreversible inhibitors bind permanently with the enzyme molecule and so effectively reduce the enzyme concentration, thus limiting the rate of reaction. Allosteric enzymes regulate metabolic pathways in cells and are made to change their shapes. They are controlled by compounds acting as non-competitive inhibitors which bind to the enzyme at sites specifically away from the active site, just as regular non-competitive inhibitors do. The only difference is that with these compounds this binding does change the shape of the enzyme which then affects the ability of the substrate to bind to the enzyme. These compounds are known as allosteric inhibitors. End-product inhibition also known as negative feedback inhibition is when the end product of a metabolic pathway selfregulates to switch off its own production as it accumulates, it acts as an allosteric inhibitor.

The time taken for the reaction to take place is generally known as the incubation time. The average rate of an enzyme catalysed reaction usually decreases as the incubation time increases, even when there is a surplus of substrate present. This decrease happens most likely, because the enzyme gradually becomes denatured with time. As the protein molecule becomes progressively disfigured, it loses its effectiveness as an enzyme. Method: 5cm3 of potato extract was placed into a boiling tube and the tube was then put into the water bath which was prepared for five minutes. 5cm3 of hydrogen peroxide was placed into another boiling tube, it was then poured into the potato extract; the tube containing the mixture was left in the water bath. The time and distance at which the foam travelled up the tube over a 2 minute, a 5 minute, and a 10 minute period were recorded. The method was then repeated for the temperatures of 35oC and 70oC. For the temperature of 0oC, the tube was placed into the ice instead of the water bath and for the temperature of 28oC the boiling tube was allowed to stand in the room.

Results: Table 1 THE DISTANCES WHICH THE FOAM TRAVELLED AT CERTAIN TEMPERATURES AFTER PARTICULAR TIME INTERVALS Temperature/ oC Distance travelled/ cm 2 mins. 4 7.5 5 0.8 0.4 Distance travelled/ cm 5 mins. 5 10 7 0.6 0.3 Distance travelled/ cm 10 mins. 6 12.5 7 0.6 0.3

0 28 r.t.p 35 70 100 Table 2:

THE VELOCITY OF THE REACTION AT VARIOUS TEMPERATURES FOR THE DIFFERENT TIME Temperature/oC Velocity of reaction Velocity of reaction (cm/min) (cm/min) 2 mins 5 mins 2 1 3.75 2 2.5 1.4 0.4 0.12 0.2 0.06 INTERVALS Velocity of reaction (cm/min) 10 mins 0.6 1.25 0.7 0.06 0.03

0 28 r.t.p 35 70 100

Discussion: The primary function of catalase enzymes in cellular metabolism is the rapid breakdown of hydrogen peroxide into water and safe oxygen compounds. Many metabolic pathways in organisms produce hydrogen peroxide as a by-product of other chemical reactions. If it were left in living tissues, this could cause extensive cellular damage that would quickly endanger the organism if peroxide accumulation continued unchecked. For this reason, catalase enzymes are necessary parts of normal cellular equipment. Catalase is found in the vast majority of living cells and nearly every aerobic cell. The equation for the break down of hydrogen peroxide by the enzyme is: 2H2O 2H2O + O2 The rate of an enzyme catalysed reaction increases with temperature until it reaches its optimum because an increase in temperature provides more kinetic energy to the molecules involved. This in turn increases the chance of enzymesubstrate complexes being formed. Enzymes are considered inactive/dormant at 0 oC and below. This factor explains why at 0 oC the distance which the foam travelled was not very high when compared or matched to the distanced travelled at 28 oC or room temperature. At r.t.p though, which was the next temperature the enzyme was tested, the foam travelled the furthest distance up the boiling tube in all of the reactions. This then proves that the rate of an enzyme catalysed reaction is directly proportional to a temperature increase before it surpasses its optimum temperature. The fact that the distance the foam travelled at r.t.p was the greatest distance seen in all of the reactions at the various temperatures says that 28 oC was the optimum temperature of the enzyme catalase. This is further proven because as the temperatures increased from 28 oC to 35 oC, 70 oC and 100 oC respectively the distance travelled by the foam decreased steadily as the temperature increased. Incubation time is the time required for the reaction to take place. Usually, as the incubation time increases, the average rate of an enzyme catalysed reaction decreases, even when there is excess substrate present. The graph results show a general and steady decrease in the amount of foam formed as the time intervals i.e. the incubation time increases. This decrease occurs because all enzymes suffer denaturation, and hence loss of catalytic activity, with time. As a result of This loss of catalytic activity the enzyme would then loses its effectiveness.

Precautions: One precaution taken was to measure the temperatures of the water baths with a thermometer to ensure that the temperatures remained constant. Another one was to ensure that the potato extract was boiled for five minutes in order to activate the enzyme. Sources of error: Parallax error could have occurred if the ruler for measuring the heights of the foam was not viewed at eye level. The results may have been affected negatively if the mixture was left in the water bath for more than the allotted time. Limitations: If adequate volumes of the potato extract or hydrogen peroxide were not added, results may have been incorrect as the reaction may have been forced to stop before it was fully completer. The water baths not remaining at constant temperature could be considered another limitation. Conclusion: Enzymes are dormant at 0oC which meant that not much activity occurred. Activity did increase though in direct proportion with temperature and hence the activity of the catalase increased at 28oC. The optimum temperature was found to be 28 oC because the activity of the enzyme decreased i.e the distance level of the foam dropped when this temperature was surpassed. This happened as a result of the enzyme becoming denatured. It was also found that enzyme activity decreases as incubation time increases.