The induced fit theory:
The induced fit theory of enzyme action is a modified version of the lock and key theory. It does not depend on such precise contact being made between the substrate and the active site. In this model, the active site is able to change its shape to enfold a substrate molecule. The enzyme takes up its most effective catalytic shape after binding with substrate. The shape of the enzyme is affected by the substrate, just as the shape of a glove is affected by the hand wearing it.
The distorted enzyme molecule in turn distorts the substrate molecule, strain or twisting the bond. This makes the substrate less stable, reduces its potential energy, and thus lowers the activation energy of the reaction. The reaction occurs and products are formed which no longer bind to the active site, and so move away. The flexible enzyme then returns to its original shape, ready to bind the next substrate molecule (see diagram next page).
Activation energy
Reactions proceed because the products have less energy than the substrates.
However, most substrates require an input of energy to get the reaction going, (the reaction is not spontaneous).
The energy required to initiate the reaction is called the activation energy.
When the substrate(s) react, they need to form a complex called the transition state before the reaction actually occurs. This transition state has a higher energy level than either the substrates or the product.
Outside the body, high temperatures often supply the energy required for a reaction. This clearly would be hazardous inside the body though! Fortunately we have enzymes that provide an alternative way with a different transition state and lower activation energy.
The rate of the reaction without any external means of providing the activation energy continues at a much faster rate with an appropriate enzyme than without it. The maximum rate that any reaction can proceed at will depend, among other things, upon the number of enzyme molecules and therefore the number of active sits available.
Factors affecting the rate of reaction
All enzymes are proteins and their functions can be affected by:
- pH – a measure of the concentration of hydrogen
Temperature:
The graph below shows how the rate of an enzyme catalyzed reaction varies with temperature. At low temperatures, the reaction takes place very slowly.
Enzymes work best at an optimum temperature.
Below this, 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, the enzymes are denatured. Bonds holding the structure together will be broken and the active site loses its shape and will no longer work.
This is because the molecules are moving relatively, substrate molecules would not often collide with the active site so the binding between substrate and enzyme therefore enzyme substrate complex is a rare event.
pH:
As with temperature, 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.
Most enzymes work fastest at a pH of somewhere around 7, that is a fairly neutral condition, however enzymes such as protease, pepsin and rennin which is found in the acidic region of the stomach, have a different pH.
Enzymes are particularly sensitive to pH because of the great sensitivity possessed by their active site. Even if a slight change in pH does not denature the enzyme, it could cause an upset in the delicate nature and chemical arrangement at the active site and so stop the enzyme from functioning.
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, 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 as shown in the graph below.
Enzyme concentration: at low enzyme concentration 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.
Cofactors
Most enzymes require additional help from cofactors, of which there are 2 main types:
1. Coenzymes - these are organic compounds, often containing a vitamin molecule as part of their structure.
Coenzymes are not permanently bound to the enzyme but may be temporarily and loosely bound for the duration of the reaction and then move away once it is completed. For example NAD, this transfers hydrogen away from one molecule in a dehydrogenase reaction and takes it to another molecule.
2. Metal ions - most speed up the formation of the enzyme-substrate complex by altering the charge in the active site e.g. amylase requires chloride ions, catalase requires iron.
Inhibitors
Inhibitors are substances that decrease enzyme activity. Inhibitors slow down the rate of a reaction. Sometimes this is a necessary way of making sure that the reaction does not proceed too fast, at other times, it is undesirable.
Inhibitors may be reversible or irreversible. Reversible inhibitors may be competitive or non-competitive
Reversible inhibitors:
Competitive reversible inhibitors: these molecules have a similar structure to the actual substrate and so will bind temporarily with the active site. The rate of reaction will be closer to the maximum when there is more ‘real’ substrate, (e.g. arabinose competes with glucose for the active sites on glucose oxidase enzyme). Non-competitive reversible inhibitors: these molecules are not necessarily anything like the substrate in shape. They bind with the enzyme, but not at the active site. This binding does change the shape of the enzyme though, so the reaction rate decreases. Non-competitive reversible inhibitors do not bind to the active site, are removed after a time, but cannot be overcome by increasing the substrate concentration.
Irreversible inhibitors: These molecules bind permanently with the enzyme molecule and so effectively reduce the enzyme concentration, thus limiting the rate of reaction, for example, cyanide irreversibly inhibits the enzyme cytochrome oxidase found in the electron transport chain used in respiration. If this cannot be used, death will occur. Some end products function to prevent a metabolic pathway continuing indefinitely. They do this by binding allosterically to the first enzyme in the pathway - acting as allosteric inhibitors. This is called end-product inhibition.
The Course of a reaction:
The reactions begin very swiftly. As soon as the enzyme and substrates are mixed, bubbles of oxygen are released quickly. A large volume of oxygen is evolved in the first minute. As the reaction continues, however, the rate at which oxygen is released gradually slows down. The reaction gets slower and slower, until it eventually slows down.
The explanation for this is quite straightforward. When the enzyme and the substrate are first mixed, there is a large amount of substrate molecules. At any moment, virtually every enzyme molecule has a substrate molecule in its active site. The rate at which the reaction will occur will depend on how many enzyme molecules there are, and the speed at which the enzyme can convert the substrate molecule into product, release it and then bind with another substrate molecule.
However, as more and more and more substrate is converted into product, there are fewer and fewer substrate molecules to bind with the enzyme. Enzyme molecules may be waiting for a substrate molecule to hit its active site. As fewer substrate molecules are left, the reaction gets slower and slower until it eventually stops.
The curve is therefore steepest at the beginning of the reaction: the rate of an enzyme-controlled reaction is always fastest at the beginning. This rate is called the initial rate of reaction.
Hypothesis
According to the trial experiment that was run, the amount of oxygen produced in the control experiment (the experiment without copper sulphate present) was observed to be about 15cm3 in the gas chamber, in the first 20seconds into the experiment, while in the real experiment (the experiment with copper sulphate present) the amount of oxygen produced in the first 20 seconds was 11cm3. 4cm3 of oxygen seemed to be short in the gas chamber when the compound copper sulphate was added.
Now, this difference could be due to environmental imbalances or it could be due to anomalous results generated during the experiment. Therefore I continued to take on readings so as to observe further trends.
Furthermore, the amount of oxygen produced in the next 40 seconds, into the control experiment was observed to be 45cm3 whilst that of its counter part, the real experiment at the same time interval was seen as 16cm3. At this moment, suspicion grew, I suspected a trend but I was a patient scientist and continued so as to observe further trends in the experimental results generated and so as to have enough raw factual evidence. Therefore, I didn’t jump into conclusions.
Totally, in the control experiment, the amount of oxygen produced at the end of 120 seconds was observed to be 56cm3. While that of the real experiment also at the end 120 seconds was observed to be 20cm3. (56-20) cm3, a difference of 36cm3 in the volume of oxygen was observed to be “missing” from the gas chamber when copper sulphate was part of the constituents of the reaction system. A difference of 36cm3 could not be an anomalous result or be due to inconsistencies.
The evidence shows that Copper sulphate is obviously doing something to the reaction system. But what might it be? It’s obviously reducing the amount of oxygen produced.
Based on the figurative evidence generated above, Copper sulphate acts as some sort of agent that reduces the amount of oxygen produced. This can only be done if the enzyme catalase is affected in some way. The enzyme catalase is what makes the oxygen production possible. Therefore I can conclude that copper sulphate acts as an agent that blocks, disturbs or better still, inhibits enzyme activity. Now, based on the background information and the analyzed preliminary results above, I would say that copper sulphate would act as an inhibitor in the breakdown of hydrogen peroxide into water and oxygen, catalyzed by the enzyme, catalase.
Prediction
When the experiment is carried out adding copper sulphate to the reactants, it will be observed that a lesser amount of oxygen will be produced than when it was done without copper sulphate within the same time limit.
Null Hypothesis
When the experiment is done, adding copper sulphate to the reactants, it will observed that a lesser amount of oxygen will “not” be produced than when it was done without copper sulphate within the same time limit.
Backup or Facts which support my prediction
Based on the background information on enzymes, we found that most enzymes, since they are protein have amino acids as their basic components or monomer. These amino acids are negatively charged ions. However, the copper in the copper sulphate molecules/compound is a positively charged ion. The common rules of magnetism states that like charges repel while unlike charges attract each other. Based on this rule, Copper sulphate and the enzyme catalase would attract each other since they both have unlike charges to each other. Therefore, the copper sulphate would more readily combine with the enzyme catalase than its substrate itself, hydrogen peroxide. Both of them bind and thus the shape of the enzyme is altered. Once the shape of the enzyme is altered, not much can happen because the function of enzymes is largely based on the three dimensional shape, the substrate enzyme activity is based on the very specific shape which has already been altered. Therefore when substrate comes in contact with disorganized enzyme, it bounces back because the shape is no longer fit for reactions to occur on, no product is formed, this generally reduces the overall amount of oxygen produced.
The enzyme, catalase contains a haem group, this is a non protein part of the enzyme called the prosthetic group of the enzyme. This group is very crucial to the function of the enzyme. The haem group contains the element iron which helps it in performing its function. When the copper in the copper sulphate molecule and the iron from the haem group of the enzyme come in contact, a reaction called a displacement reaction occurs, iron displaces copper from its compound in the reaction because it is higher up in the reactivity series of metals, thereby forming iron sulphide. While the copper stays in place of the iron in the haem group, i.e. the active site of the enzyme. As we have seen in the background information, the enzyme molecules function greatly depends on its shape and chemical arrangement, by this reaction occurring, the chemical arrangement of the enzyme is changed and so cannot function properly and therefore reduces the amount of oxygen it produces.
Again, a similar reaction scenario occurs. The active site of catalase contains a chemical element, magnesium. Magnesium is very high in the reactivity series of metals. When magnesium and copper in copper sulphate are in the same reaction, a similar displacement reaction occurs. Magnesium displaces copper from the copper sulphate and forms magnesium sulphate, while copper is left to bind in the active site of enzymes, catalase. This again causes an element distortion, which destroys the enzyme structure and prevents it from performing its function the way it should and consequently, reduce the total rate of activity of the enzyme to the minimum or stop it completely.
As may have notice in the background information, inhibitor molecules are molecules that are very similar in size shape and structure to structure to substrate molecules because they bind in place of the substrate molecules in the active site, which is very sensitive to the substrate in which it combines with. An enzyme would not bind with a substrate which does not have a complementary shape to its active site, in the same way; it will not bind with a substrate with no complementary shape.
If another molecule (Inhibitor molecule) binds in place of the substrate (hydrogen peroxide in the active site of catalase, that molecule needs to have the same structure as that of hydrogen peroxide.
If the compound copper sulphate acts as an inhibitor in the reaction, then its structure would be very similar to that of hydrogen peroxide.
Copper sulphate is a compound composed of two molecules, an element, copper and a radical sulphate. The element, copper has a charge of +2 while the radical has a charge of 2-, together they cancel out and make zero to make the compound a stable one, a neural compound.
Again, hydrogen peroxide is composed of 2 elements, hydrogen and oxygen. Hydrogen has a charge of +1, and since there are two of the hydrogen atoms; they all form a charge of +2. Oxygen also has a charge of -1 in this compound, also there are two of this therefore they naturally make a charge of -2.This also balances the overall charge making it zero and neutral compound. Therefore these two molecules are very similar indeed. Based on the undisputed facts presented above, I think it is quite clear that the compound copper sulphate would act as an inhibitor in the break down of hydrogen peroxide into its components.
Method of Experiment
Apparatus
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gas-syringe(100cm3)
- conical flask
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2cm3 syringe
- rubber pipe
- rubber bung
- Pipette and pipette filler
- stop watch
- clamp stand
- table mat
Reagents
- Yeast (catalase)
- Hydrogen peroxide
- Water
- copper sulphate
Uses of apparatus during Experiment
Gas-syringe (100cm3): Attached directly to the conical flask by a rubber tube, for collecting and measuring the exact amount of Oxygen given off directly from the beaker during the experiment.
Clamp stand: Made up of the head, boss, stand and clamp. Generally for support i.e. for holding and stabilizing the gas syringe in one position during the experiment.
Conical Flasks: This is the reactions described above takes place. It contains the substrate, Hydrogen peroxide, the source of catalase, yeast, water, to vary the concentrations and finally the copper sulphate, the variable in concern. Attached directly to the gas syringe for measurement, this is part of the fair test.
Stop Watch: For measuring the total time for the experiment (120seconds) and the time intervals in which the amount of oxygen produced would be measured. (20 seconds).
Pipette and pipette filler: These are used to accurately measure the volumes and concentrations of the substrate (H202) and the variable in concern, copper sulphate.
Rubber pipe: The rubber connection is used for the transfer of the oxygen produced straight forwardly from the reaction into the gas syringe for measurement and recording, without any medium of barrier.
2cm3-Syringe: To accurately measure the amount of yeast extract (source of catalase) this is added to the reaction for powering.
Rubber bung: this is used to cork the flask to prevent the oxygen gas from escaping.
Table Mat: This is what is used to support the whole experimental setup shown above, for stability and therefore so as to generate more accurate results.
Procedures
Without copper sulphate (control)
- Rinse out all equipment which would be used to set up apparatus.
- Prepare volumes to be used according to the dilution table.
- Set up the apparatus as shown in the diagram above.
-
Measure out 1st concentration of hydrogen peroxide and put into conical flask.
- Measure out required amount of water needed to keep a constant overall volume.
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Measure out 2cm3 of yeast and inject into conical flask.
- Record the volume of oxygen evolved at every 20 second interval in the result table.
- Repeat for the different concentrations of hydrogen peroxide.
- Record volume of oxygen evolved at every 20 second interval in the results table.
- Take three readings for each concentration to ensure accuracy.
Dilution Table of Reactants without Copper Sulphate
Conc. means concentration
With copper sulphate
- Rinse out all equipment which would be used to set up apparatus.
- Prepare volumes to be used according to the dilution table below.
- Set up apparatus as shown in the diagram above.
-
Measure out 1st concentration of hydrogen peroxide and put into conical flask.
-
Measure out 2cm3 of copper sulphate and put into conical flask.
- Measure out required amount of water needed to keep a constant overall volume.
- Record the volume of oxygen evolved at every 20 second interval in the result table.
-
Repeat for the different concentrations of hydrogen peroxide using 2cm3 of copper sulphate and adding the required amount of water to keep the overall volume constant.
- Record volume of oxygen evolved at every 20 second interval in the results table.
- Take three readings for each concentration to ensure accuracy.
Dilution Table of Reactants with Copper Sulphate
Conc. means concentration
Safety/ risk assessment
When carrying out this experiment I made sure;
- I wore safety goggles to protect my eyes from hydrogen peroxide
- All apparatus was kept away from the edge of the desk to prevent breakages
- A lab coat was worn to prevent the corrosive hydrogen peroxide from damaging my clothes
- All chairs were tucked in and I avoided moving about
I also made sure I took my risk assessment form (attached) into consideration.
Justification for Use of Apparatus
Apparatus ‘i’ seemed to have some complications during the set-up and organization, even before the experiment started. It also seemed to lack the ability of apparatus ‘ii’ during oxygen collection and recording of experimental results due to very complicated principles of gas upward delivery i.e. gas displacing water.
The principle was that any gas such as oxygen was denser than water and it would displace water upwardly and take its place at the very top, therefore, simple calculations of subtraction had to be done before the volume of oxygen could be known, during short intervals of 20 seconds which made them seem complicated and inaccurate. Whereas, apparatus ‘ii’ used a very simple principles of just measuring the amount of oxygen produced in a gas chamber and all the person carrying the experiment had to do was measure the amount in the table provided. Therefore, apparatus ‘ii’ was easier to use based on the preliminary experiment and the four criteria below:
- Apparatus ‘ii’ was very compact, very easy to use and setup because there were less materials and equipments involved.
- Due to simple principles of apparatus ‘ii’ the experiment was very easy to carry out
- No water was involved in the set-up and experimental principles of apparatus ‘ii’ therefore, the experiment was very easy to carry out, no spills or messes were generated before the experiment was started.
- The readings were very easy to take in apparatus ‘ii’ unlike apparatus ‘i’ where the principle of oxygen gas displacing water upwardly, then the water level before displacement had to be subtracted from the oxygen level after displacement before the amount of oxygen evolved could be known, over a very limited time interval of 20 seconds, the readings of apparatus ‘ii’ could be taken directly in the gas chamber. No complications of subtractions over a very small time interval involved.
- Apparatus ‘ii’ took very little time to set-up unlike apparatus ‘i’ which took very little time due to enormous equipments and the water involved.
- Readings of Apparatus “ii” could be taken directly and more easily because the product, oxygen gas, was collected directly into the gas chamber with very vivid lamentations.-Direct measurement.
- During the process of experiment “i”, oxygen bubbles which were evolved from the reaction, often get delayed at the edge , inside the water in the measuring cylinder due to the complicated principles, therefore not the displacing the water it is suppose to displace and consequently the reading is not counted because the water isn’t displaced by the gas.
- Apparatus ‘ii’ could easily be set-up again if things went wrong during the experiment and one had to start again.
Due to the justifications above, apparatus ‘ii’ was in a better position to produce the precise and accurate results that is required by the experiment.
Table of results with copper Sulphate
Table of results without copper sulphate
The table of results as seen above is the table provided with coordinated spaces where all the readings of oxygen produced, as observed in the gas syringe at the intervals of 20 seconds over the range of 140 seconds would be recorded, so as to observe trends, similarities and differences in the raw data recorded, this is where reference would be made to later in the analysis, so as get to a conclusion.
For fair test sake, each reading from the experimental apparatus has to be taken three times and later; an average result would be reached by adding up the three readings and dividing them into three. This average is the figure that would be used for further calculations in the further stages of this investigation; this is so as to trade off errors and to minimize the errors that are generated during the experiment by performing them more than once.
Bibliography
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RICHARD FOSBERY, MARY JONES, DENNIS TAYLOR- BIOLOGY 1
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ANN FULLICK-BIOLOGY SECOND EDITION
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CJ CLEGG WITH DG MACKEAN – ADVANCED BIOLOGY PRINCIPLESAND APPLICATION.
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CHRIS LEA, PAULINE LOWRIE- ADVANCED LEVEL BIOLOGY
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RICHARD FOSBERY, JENIFFER GREGORY, IANTO STEVENS-BIOLOGY 1
