Introduction
In this experiment I will attempt to investigate how the change in temperature effects the catalyse reaction and what the optimum temperature is.
Key factors
Scientific knowledge
Enzymes:
Because enzymes are proteins they can be destroyed at high temperatures, this is called denaturing. Enzymes are used to catalyse (speed up) chemical reactions. There are many types of enzymes and they are all used to break down certain food molecules, this is described in the lock and key theory and the induced fit theory.
Enzyme theory:
Lock and key theory.
Enzymes are biological catalysts. The lock and key theory was suggested in 1894 by Emil Fischer and properly described as follows "The specificity of an enzyme (the lock) for its substrate (the key) arises from their geometrically complementary shapes".
The lock and key theory is simply a way of describing how specific an enzyme is for its substrate. Just like a lock requires a specifically shaped key for it to work so does an enzyme. Each enzyme is a protein which is a polypeptide chain folded into a complex 3 dimensional structure. Part of that structure contains the active site which is where the enzyme can bind to the substrate on which it will perform some chemical reaction. Because each enzyme performs a specific task on a specific substrate the active centre of the enzyme can be considered to be the "lock" which requires the specific "key" or substrate to perform the function. Smaller keys, larger keys, or incorrectly positioned teeth on keys (incorrectly shaped or sized substrate molecules) do not fit into the lock (enzyme). Only the correctly shaped key (substrate) opens a particular lock. If we imagine the enzyme as the lock and the substrate the key - the key is inserted in the lock and if this is his right enzyme for the substrate the lock is turned, and the door is opened and the reaction proceeds.
The active site is the specific region of the enzyme which combines with the substrate. The products are released from the enzyme surface to regenerate the enzyme for another reaction cycle.
The active site has a unique geometric shape that is complementary to the geometric shape of a substrate molecule. This means that enzymes specifically react with only one or a very few similar compounds.
Induced Fit Theory:
This theory uses instead of the analogy the key in the lock but instead the glove in the hand this theory also explains that a protein is flexible. Enzymes act as ...
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The active site is the specific region of the enzyme which combines with the substrate. The products are released from the enzyme surface to regenerate the enzyme for another reaction cycle.
The active site has a unique geometric shape that is complementary to the geometric shape of a substrate molecule. This means that enzymes specifically react with only one or a very few similar compounds.
Induced Fit Theory:
This theory uses instead of the analogy the key in the lock but instead the glove in the hand this theory also explains that a protein is flexible. Enzymes act as biological catalysts. They are globular proteins that have a specific shape within which there is a functional portion known as the active site. Enzymes lower the activation energy of a reaction, allowing it to proceed at a lower temperature than it would normally. In an enzyme controlled reaction, the general term for the substance on which the enzyme acts is substrate and the substances formed at the end of there action are known as the products. The enzyme molecule and the substance it acts on fit together very precisely, giving rise to the name lock and key theory of enzyme action. In practice, the enzyme is thought to change shape slightly and so mould itself to the shape of the substance it acts on. This is called the induced fit theory of enzyme action
Structure and Function of an Enzyme.
Enzymes are large proteins that speed up chemical reactions. In their spherical structure, one or more polypeptide chains twist and fold, bringing together a small number of amino acids to form the active site, or the location on the enzyme where the substrate binds and the reaction takes place.
Enzyme and substrate fail to bind if their shapes do not match exactly. This ensures that the enzyme does not participate in the wrong reaction. The enzyme itself is unaffected by the reaction. When the products have been released, the enzyme is ready to bind with a new substrate.
Properties of Enzymes
As the Swedish chemist Jöns Jakob Berzelius suggested in 1823, enzymes are typical catalysts: they are capable of increasing the rate of reaction without being consumed in the process.
Some enzymes, such as pepsin and trypsin, which bring about the digestion of meat, control many different reactions, whereas others, such as urease, are extremely specific and may accelerate only one reaction. Still others release energy to make the heart beat and the lungs expand and contract. Many facilitate the conversion of sugar and foods into the various substances the body requires for tissue-building, the replacement of blood cells, and the release of chemical energy to move muscles.
Pepsin, trypsin, and some other enzymes have in addition, the peculiar property known as autocatalysis, which permits them to cause their own formation. As a consequence, these enzymes may be reproduced in a test tube.
As a class, enzymes are extraordinarily efficient. Tiny quantities of an enzyme can accomplish at low temperatures what would require violent reagents and high temperatures by ordinary chemical means. About 30g of pure crystalline pepsin, for example, would be capable of digesting nearly 2 metric tons of egg white in a few hours.
Each enzyme is selectively specific for the substance in which it causes a reaction and is most effective at a temperature peculiar to it. Although an increase in temperature may accelerate a reaction, enzymes are unstable when heated. Many enzymes require the presence of another ion or a molecule called a cofactor, in order to function.
As a rule, enzymes do not attack living cells. As soon as a cell dies, however, enzymes that break down protein rapidly digest it. The resistance of the living cell is due to the enzyme's inability to pass through the membrane of the cell as long as the cell lives. When the cell dies, its membrane becomes permeable, and the enzyme can then enter the cell and destroy the protein within it. Some cells also contain enzyme inhibitors, known as anti-enzymes, which prevent the action of an enzyme upon a substrate.
Enzyme reactions
An enzyme-catalysed reaction, the rate is usually expressed in the amount of product produced per minute. The energy barrier between reactions and products governs reaction rate. In general, energy must be added to the reactants to overcome the energy barrier. This added energy is termed "activation energy", and is recovered as the reactants pass over the barrier and descend to the energy level of the products. Enzymes can accelerate the rate of a reaction. Catalysts accelerate the rates of reactions by lowering the activation energy barrier between reactants and products. All chemical reactions speed up as the temperature is raised. As the temperature increases, more of the reacting molecules have enough kinetic energy to undergo the reaction.
Enzyme classification
Enzymes are classified into several broad categories, such as hydrolytic, oxidising, and reducing, depending on the type of reaction they control. Hydrolytic enzymes accelerate reactions in which a substance is broken down into simpler compounds through reaction with water molecules. Oxidising enzymes, known as oxidises, accelerate oxidation reactions; reducing enzymes speed up reduction reactions, in which oxygen is removed. Many other enzymes catalyse other types of reactions.
Individual enzymes are named by adding ASE to the name of the substrate with which they react. The enzyme that controls urea decomposition is called urease; those that control protein hydrolyses are known as proteinases. Some enzymes, such as the proteinases, trypsin and pepsin, retain the names used before this way of naming was adopted. Enzymes are large proteins that speed up chemical reactions. In their round structure, one or more polypeptide chains twist and fold, bringing together a small number of amino acids to form the active site, or the location on the enzyme where the substrate binds and the reaction takes place. Enzyme and substrate fail to bind if their shapes do not match exactly. This ensures that the enzyme does not participate in the wrong reaction. The enzyme itself is unaffected by the reaction. When the products have been released, the enzyme is ready to bind with a new substrate.
Primary structure
AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11-AA12 etc
This structure twists and turns because of the R groups in the amino acids. Every R group is different
If you zoom in on one of the amino acids and look at the structure you will see:
H H R
NH3 - C – C – C - CooH
H H R
Because of the attraction between the r groups weak hydrogen bonds are formed, these bonds are what hold the structure together and if the enzyme its alpha helix or beta sheet shapes.
Measuring enzyme reactions
The two ways in which an enzyme reaction can be measured they are rate of reaction and time course. Time course reactions are usually plotted by measuring either the formation of products or the disappearance of the substrate. If the temperature is increased the rate of an enzyme reaction will rise/increase up to a point at which its molecular structure is disrupted. At this point the enzyme is said to be denatured. With a fixed amount of enzyme the addition of more substrate will cause the rate of reaction to increase until all the enzyme molecules are being used. At this point the rate of reaction levels off because the enzyme is limiting the reaction. An increase in the amount of enzyme will cause a proportional increase in the rate of reaction provided that there is excess substrate. Enzymes work in a narrow range of pH outside of which the hydrogen bonds between the NH and CO groups are broken. A solution that prevents changes in pH is called a buffer solution.
Scientific knowledge used to plan
Structure in enzymes
The different levels of protein structure are known as primary, secondary, tertiary, and quaternary structure. There are 20 common amino acids are classified by their functional group, or their "R" group. When the weak hydrogen bonds that help the enzyme take its shape break because of the heat the enzymes have become denatured.
Primary structure
The primary structure is the sequence of amino acids that make up a polypeptide chain. 20 different amino acids are found in proteins. The exact order of the amino acids in a specific protein is the primary sequence for that protein.
AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11-AA12 etc
This structure twists and turns because of the R groups in the amino acids. Every R group is different
If you zoom in on one of the amino acids and look at the structure you will see:
H H R
NH3 - C – C – C - CooH
H H R
Because of the attraction between the r groups weak hydrogen bonds are formed, these bonds are what hold the structure together and if the enzyme its alpha helix or beta sheet shapes.
Secondary structure
The amino acids form regular repeating patterns folding along the protein back bone.
There are two common structures, the alpha helix and the beta pleated sheet.
Alpha Helix
In an alpha helix, the polypeptide backbone coils around an imaginary helix axis in clockwise direction to get its shape. The most common structure is the alpha helix. This structure gives stability to the unit because there is a weak hydrogen bond between the various peptide bonds. Notice that the left handed helical backbone is made up of the peptide chain. The R groups point out from the structure at a 90 degree angle.
Beta sheet
In the beta sheet secondary structure, the polypeptide backbone is nearly completely strait. Beta sheets are a combination of two or more beta strands. The strands are held together and stabilized by hydrogen bonding. There are two types of sheets: either parallel or anti parallel depending on the orientation of the peptide chain.
Tertiary structure
Tertiary structure refers to the overall folding of the entire polypeptide chain into a specific 3D shape. The tertiary structure of enzymes is often a compact, globular shape.
Quaternary structure
Many proteins are formed from more than one polypeptide chain. The quaternary structure describes the way in which the different subunits are packed together to form the overall structure of the protein.
How will the enzyme structure help me plan?
Knowing the enzyme structure will help me plan my experiment because it will help me understand how an enzyme becomes denatured and that will help me to plan my experiment because I will then know which temperatures will be the best to use.
Plan of procedure
To do this experiment I will:
- Set each water bath to the right temperature because if I didn’t my results would be inaccurate because yeast is very receptive to change so I would be able to see that change in my results. I will need to use a thermometer to measure the temperature to make sure the temperature is right.
- Measure out 2 cm3 of yeast and hydrogen peroxide, using a pipette to get the exact measurement and I will also use a small thin measuring cylinder. I will also need to make sure each of the measuring cylinders is clean and dry. If they weren’t clean and I added hydrogen peroxide to a dirty measuring cylinder which had yeast in the reaction would already start to happen this wouldn’t make it a fair test and it would alter my results table. If the measuring cylinder had water or any other liquid in it would lower the concentration of the hydrogen peroxide because of that my results table would be inaccurate .If I didn’t have the exact amount of yeast and hydrogen peroxide my results would be inaccurate because the froth height would be higher or lower because there is more of the yeast and hydrogen peroxide.
- Place a thermometer into the hydrogen peroxide and the yeast, so I know when the yeast or hydrogen peroxide has reached the exact temperature. I need to know the exact temperature because the yeast is very receptive to change so if the temperature was higher or lower I may get anomalous results.
- Place the yeast and hydrogen peroxide into the water bath making sure that no water enters the measuring cylinders because if it did the concentration of the yeast and hydrogen peroxide would be lowered and if that happened would have anomalous results.
- When the yeast has reached the needed temperature (I will know this by reading the thermometer) place either one of the liquids into another measuring cylinder and then pour the other liquid into that measuring cylinder and measure the max froth height by using the markers on the measuring cylinder. If it exceeds the maximum height place a ruler against the highest marking on the measuring cylinder, this may not be accurate but it is the only way of measuring it as a bigger beaker doesn’t have as many markings on it. I will be measuring the maximum froth height reached because I think this is the best way of measuring the maximum amount of oxygen produced.
- After measuring the maximum froth height I will note the results then I will repeat the temperatures I have just done twice to make sure that my results were not anomalous.
- I will repeat these steps for all of the temperatures.
- I will then average out the results.
Range of observations
I will repeat all of these temperatures twice to make sure that none of my results are anomalous.
For this experiment I will be doing the temperatures:
- 0 oc
- 10 oc
- 20 oc
- 30 oc
- 40 oc
- 50 oc
- 60oc
After repeating the results twice I will look at my results and if any are anomalous then I will repeat the experiment. After getting rid of any anomalous results I will average out the results. I will also use other group’s results to check my results to see if any of the results are anomalous.
Precision
I have decided to use the following equipment:
- measuring cylinders (10cm3)
- water baths
- ice baths
- pipettes
- Paper towels
- Ruler
- Thermometer
I used a small measuring cylinder to measure out the amounts of hydrogen peroxide and the yeast because in a smaller measuring cylinder I can achieve more accurate results because there are more detailed markings so I will know if I have done 2cm3 or 1.9cm3.
I am going to use water baths to keep the water at a certain temperature. I think that using a water bath to keep the water heated is the best apparatus to use for certain temperatures because a water bath can keep the water at a specific temperature for a long time whereas a beaker of water heated by a Bunsen burner can begin to lose heat and when you try to reheat the water you could go above the required temperature. You could also go above the required temperature
I will be using an ice bath to keep the temperature at 0oc. I think that an ice bath would be the best apparatus to use for this temperature because I couldn’t use a water bath because that would keep the temperature above o and a Bunsen burner would keep the temperature above 0 oc.
I will use a small pipette to add more of the liquid into the measuring cylinder because then if I need to add 1 more drop of a liquid into a measuring cylinder it would be a lot easier than pouring it strait from the bottle and it works the other way round as well because if I have added to much liquid to the measuring cylinder I can take out small amounts of the liquid using the pipette.
I will be using paper towels to dry the measuring cylinders. I think that using a paper towel is the best thing to use when drying a measuring cylinder because a paper towel can be twisted and pushed into the measuring cylinder to dry it out.
If the froth exceeds the maximum height on the measuring cylinder I will use a ruler to measure how high the froth has gone. I think that a cm ruler will be the best thing to use because 1cm on the ruler is the same height as 1cm3 on the measuring cylinder.
I will be using a thermometer to measure the temperature of each of the liquids; I will be using a long thin thermometer to get an accurate reading. I think that using a thermometer will be the best apparatus to because a thermometer can give accurate readings and when a longer thinner thermometer is used it can give more accurate readings because I can get decimal point answers.
Justified prediction
For this experiment I think that the froth height will be at its highest at 40 oc because that it the temperature that is closest to body temperature. I know that enzymes work best at body temperature because the human body is very well designed to make sure that everything works well and if the enzymes and other body functions worked better at higher or lower temperatures then the body temperature would change to have better conditions for the enzymes to work so I think that the optimum temperature will be 40oc. I predict that the enzyme will become denatured, and therefore will work at a slower rate after 40 - 45°C. I think the reason for this prediction is because every enzyme has a temperature range of optimum activity. Outside that temperature range the enzyme is rendered inactive. This occurs because as the temperature changes enough energy is supplied to break some of the molecular bonds. When these forces are disturbed and changed the active site becomes altered in its ability to accommodate the substrate molecules it was intended to catalyse. Most enzymes in a human body shut down beyond certain temperatures. This can happen if body temperature gets too low (hypothermia), or too high (hypothermia).
From my background knowledge it is evident that as temperature increases, the rate of reaction also increases. However, the stability of the protein also decreases due to thermal degradation. Holding the enzyme at a high enough temperature for a long period of time may cook the enzyme.
I think that the froth height will not be very high at the temperature 0 oc in-fact it will be the lowest out of all of the temperatures because I know that enzymes work best at around 37-39oc because that is body temperature and 0 oc it way below that temperature so I think that the froth height for 0 oc will be around 6-10cm3.
I also think that when I do the next temperature in my experiment which is 10 oc higher the froth height will be higher until it reaches 40 oc because the after that the enzymes begin to denature because of the extreme temperatures the weak hydrogen bonds which the primary structure of amino acids have formed begin to break.
I predict that that if it took 50 seconds for the froth to reach the 10cm3 mark then at 20 oc it would take 40 seconds for it to reach the 10cm3 mark and the time taken for it to reach the 10cm3 mark will take a smaller amount of time, each time the temperature increased until it reached 40oc because the heat speeds up the reaction. My prediction is supported by Kinetic Theory in that if I apply twice as much heat there will be twice as much particle vibration therefore the reaction will happen twice as quickly. It is also backed by Collision Theory in that if I apply twice as much heat there will be twice as many collisions and therefore the rate of reaction will double. This will only be so until the enzyme denatures after its optimum temperature
Since enzymes are catalysts for chemical reactions, enzyme reactions also tend to go faster with increasing temperature. However, if the temperature of an enzyme catalysed reaction is raised still further, an optimum is reached: above this point the kinetic energy of the enzyme and water molecules is so great that the structure of the enzyme molecules starts to be disrupted. The positive effect of speeding up the reaction is now more than offset by the negative effect of denaturing more and more enzyme molecules. Many proteins are denatured by temperatures around 40 - 50°C, but some are still active at 70 - 80°C, and a few withstand being boiled. So, my first prediction is that the enzyme will become denatured at around 40°C, and secondly, that as the temperature increases the reaction rate will increase by 50%, due to the molecules colliding together at a higher speed (kinetic theory) due to their extra energy obtained by the increase in temperature. My prediction is supported by Kinetic Theory in that if I apply twice as much heat there will be twice as much particle vibration therefore the reaction will happen twice as quickly. It is also backed by Collision Theory in that if I apply twice as much heat there will be twice as many collisions and therefore the rate of reaction will double. This will only be so until the enzyme denatures after its optimum temperature: 45°C.
On the next page there is a graph of what I think the actual graph of the results is going to look.
From the graph you can see that the maximum froth height rises until it reaches its optimum temperature (40 oc) then the graph starts to fall.
Secondary source data
In this experiment I intend to use at least 1 other piece of data to check my results against I am also going to use a set of results from a computer program called focus education software. In total I am using 3 sets of data including mine.