Enzymes

Question 1

Define a catalyst

Answer 1

Chemical that speeds up the rate of a reaction by providing an alternative pathway which lowers the activation energy of the reaction and remains unchanged and reusable at the end of the reaction.

Question 2

Define an enzyme

Answer 2

A protein molecule made by cells that acts as a catalyst and increases the rate of a chemical reaction by lowering the activation energy of the reaction .

Question 3

Explain what are enzymes

Answer 3

1) Enzymes are biological catalysts.

2) Their actions affect both the structure and function within cells, tissues and organs.

3) They speed up reactions that would otherwise require high temperatures, high pressures, extremes of pH and high concentrations of reactants – all factors that would kill the organism. They can speed up metabolic reactionsby up to 10^12times.

Question 4

Explain the role of enzymes

Answer 4

1) Life is completely dependent on enzymes.

2) Enzymes are more specific than chemical catalysts and so do not produce
unwanted by-products and rarely make mistakes.

3) The cells in which enzymes are made and/or act can also regulate their production and activity to fit the needs of the cell or organism at that time.

4) Enzymes can only increase the rates of reaction up to a certain point. This is
called the Vmax.

5) Enzymes form the biological molecules (anabolic reactions) that make up living tissues e.g. production of collagen.

Enzymes break down biological molecules (catabolic reactions) that provide organisms with energy and building materials e.g. respiration.

Question 5

Explain the sites of activity of enzymes

Answer 5

All enzymes are proteins and so are made in the cells by protein synthesis.

Most enzymes remain inside the cells, but many cells are specialised to enzymes that are secreted to work outside cells in the environment or inside a body cavity such as gut or blood.

Enzymes catalyse a wide range of intracellular and extracellular reactions.

Question 6

Define intracellular and extra cellular reactions and provide an example

Answer 6

Intracellular enzymes: Enzymes that catalyse reactions within cells e.g. Catalase

Extracellular enzymes: Enzymes that catalyse reactions outside cells. e.g. Amylase and trypsin

Question 7

Explain what are intracellular enzymes

Answer 7

Intracellular enzymes can be:

a) Free in solution, e.g. in the cytosol, nucleoplasm, mitochondrial matrix and the stroma of chloroplasts.

b) Fixed in place, e.g. on either side of the cell surface membrane and in the inner membranes of mitochondria and chloroplasts.

Over 1000 metabolic reactions can be taking place inside a cell at the same time, each being catalysed by a different enzyme.

Some of these reactions are part of a metabolic pathway. A metabolic pathway is a series of consecutive reactions, with the use of specific enzymes in certain steps. The reactants, intermediates and products are known as metabolites.

Question 8

Explain an example of an intracellular enzyme (catalase)

Answer 8

1) Catalase is an enzyme found in both animal and plant tissue that works inside cells to catalyse the breakdown of hydrogen peroxide (a waste product of many metabolic processes in cells).

2) Catalase consists of four polypeptide chains and a haem group with iron.

3) Hydrogen peroxide is a powerful oxidising agent and so very toxic so needs to be removed/destroyed fast.

4) Without the help of catalase, hydrogen
peroxide would take months to degrade.

5) Some cells actually use hydrogen peroxide to kill pathogens, cells infected with viruses and cancer cells.

6) It is the fastest-acting enzyme, turning over about 6 million reactions per second.

Question 9

Explain what are extracellular enzymes

Answer 9

Extracellular enzymes catalyse reactions that occur outside the cells, such as those involved in digestion .

They are secreted from the cells where they are made and act on their substrates, extracellularly.

Question 10

Explain an example of an extracellular enzyme (amylase/trypsin)

Answer 10

Amylase:
1) Amylase is produced in the
salivary glands.

2) It acts in the mouth to digest the
polysaccharide starch to the disaccharide
maltose.

3) It is also made in the pancreas, and
acts in the small intestine.

Trypsin:

1) Trypsin is made in the pancreas.

2) It acts in the lumen of the small intestine to digest proteins into smaller peptides by hydrolysing peptide bonds.

3) Its optimum pH is between 7.5 and 8.5.

Question 11

Explain enzyme structure in relation to the active site

Answer 11

1) Enzymes are large globular proteins with a specific area, an indentation or cleft on the surface of the molecule, called an active site.

2) Enzyme specificity is determined by the
shape of its active site. The degree of specificity varies; some are highly specific to one reaction, others are less specific and catalyse a number of reactions of the same type, e.g. protease.

3) The active site consists of around 6 to 10
amino acids and the tertiary structure is crucial.

4) The features of the active site and
the type of substrate it accepts are
determined by the R groups of these
amino acids.

Question 12

What’s is an enzyme-substrate complex

Answer 12

The complex that forms in the active site after the reaction is complete but before the product or products leave.

Question 13

What is activation energy

Answer 13

In a chemical reaction, the activation energy is the minimum amount of energy required before the reaction will start.

Enzymes reduce the amount of activation energy needed so reactions can often happen at lower temperatures. This speeds up the rate of reaction.

Question 14

Explain anabolic reactions

Answer 14

In anabolic reactions, the enzyme holds the substrates close to one another, reducing repulsion and allowing them to bond more easily.

Question 15

Explain catabolic reactions

Answer 15

In catabolic reactions, fitting the substrate into the active site puts strain on the bonds, making them easier to break.

Question 16

What is the lock and key hypothesis

Answer 16

The idea that an enzyme’s active site is complementary in shape to the substrate in the same way as a key (substrate) and lock (enzyme).

Question 17

Explain the problems with the lock and key hypothesis

Answer 17

a) The lock and key theory does not fully explain how enzyme and substrate molecules are able to collide successfully and form enzyme-substrate complexes.

b) The collisions are random and so it would be quit difficult for the enzyme and substrate to collide in the correct way for the substrate to bind with the active site.

c) This led to the formation of a modified
version of the lock and key hypothesis
called the induced-fit hypothesis by Daniel
Koshland in 1958.

Question 18

What is the induced fit hypothesis

Answer 18

The theory that the active site of an enzyme changes shape during the binding of a substrate molecule, and this puts strain on the substrate molecule contributing to the reaction.

Question 19

Explain the induced fit hypothesis

Answer 19

a) When the substrate molecules fit into the enzyme’s active site, the active site changes shape slightly to mould itself around the substrate molecule.

b) The active site still has a complementary shape to the substrate, but on binding, the subtle changes of the R groups of the amino acids provide a more precise conformation that exactly fits the substrate. This allows a more effective binding.

Question 20

Explain an enzyme-produce complex

Answer 20

Enzyme product complexes are formed towards the end of the reaction. Just before the reaction is complete, the substrate is changed into a product, which remains bound to the enzyme before it is released. We call this the “enzyme-product complex”.

Most enzyme reactions are reversible. The importance of the enzyme-product complex is that in most cases, the enzyme could induce the product to change back into the substrate, i.e. reverse the reaction. This is why most reactions catalysed by an enzyme are reversible.

Question 21

Explain the structure of enzymes

Answer 21

Enzymes usually have an almost spherical shape. Enzymes can be large, or they can be compact, depending on the substrates to which they bind.

Enzymes are usually soluble in water. This means that they have a large number of polar (hydrophilic) amino acids in their polypeptide chains.

All enzymes have an active site with a specific shape. The active site of an enzyme binds to a substrate (the target). The structure of an enzyme’s active site determines which substrates it is capable of binding to.

Question 22

Explain how enzyme structure relates to their function and how mutation can affect enzyme activity

Answer 22

Enzymes are similar to most proteins. Enzymes are proteins and therefore their chemical properties are more or less similar to most proteins. The majority of the properties that will be discussed in this section can be readily applied to proteins as well.

Like proteins, enzymes derive their properties from their tertiary structure. Changes to their tertiary structure will lead to changes in their functionality. The tertiary structure of an enzyme determines the structure of its active site, and therefore its substrate binding ability.

Mutations can disrupt enzymes. Mutations in the DNA of an organism can lead to the development of proteins and enzymes with mutations. These mutations can cause a protein or enzyme to lose its intended function.

Question 23

Explain the mechanism of enzymes forming products

Answer 23

Substrate binds to the enzyme’s active site. As substrate binds, the shape of the active site changes slightly an enzyme-substrate complex is formed. If the substrate enables the active site’s shape to change in the right way then the reaction takes place and an enzyme-product complex is formed. The products are then released from the active site.

Question 24

What factors affecting enzyme activity

Answer 24

1) Temperature

2) pH

3) Substrate concentration

4) Enzyme concentration

Question 25

What is the optimum temperature

Answer 25

The temperature at which the rate of a process is at its maximum. Above or below this temperature, the rate of reaction is lower. The optimum temperature varies according to the habitat to which an organism is adapted.

Question 26

Explains the effect of temperature on enzyme activity

Answer 26

1) Enzyme and substrate molecules move continually and randomly as a result of their kinetic energy. 2) An increase temperature increases the kinetic energy of the molecules; the molecules will move faster, increasing the chances of collisions. There will be more successful collisions as a result and so more ESC and more product will be formed. This causes an increase in the reaction rate. 3) The rate of reaction continues to increase until the enzyme reaches its optimum temperature. 4) At temperatures higher than the optimum, the enzyme loses its stability and begins to be disrupted. 5) As the temperature rises, the movement of the molecules increases and causes the molecules to vibrate. This can break the weaker hydrogen bonds and ionic bonds that stabilise the tertiary structure of the enzyme, disrupting the specific shape of the active site. The enzyme is now denatured.

Question 27

Explain what is denaturation

Answer 27

A change in the tertiary structure of proteins such as enzymes, which means it no longer functions. Thermal denaturation is usually irreversible, denaturation by changes in pH is often reversible.

Question 28

What is the temperature coefficient

Answer 28

The ratio between the activities of a process, such as an enzyme-catalysed reaction, at two different temperatures. If the two temperatures are 10°C apart, then the temperature coefficient is written as Q10.

Question 29

How can you calculate the temperature coefficient

Answer 29

rate of reaction at (x + 10)°C ————————————— =Temperature coefficient rate of reaction at x °C Where X = any chosen temperature

Question 30

Explain the affect of pH on enzyme activity

Answer 30

1) pH is a measure of the concentration of hydrogen ions (H+) in solution. The pH scale is a logarithmic scale, which means that a change from pH 6 to 7, is equal to a change in H+ concentration of x10. 2) Enzymes function over a particular range of pH. Above and below the optimum pH, the enzyme performs less well, and beyond the extremes of the range, the enzyme becomes denatured. 3) The optimum pH is specific for each enzyme, some enzymes will work across a wide range of pH, others have a very narrow range of pH that they will function across.

Question 31

Explain how changes in pH affect bonds

Answer 31

1) Excess hydrogen ions will interfere with the hydrogen bonds and ionic forces that hold the tertiary structure of the protein together. This causes the protein (enzyme) to change shape, in the case of enzymes causing the active site to change shape. 2) Increasing H+ concentration will also alter the charges on the active site of enzyme molecules, as more protons will cluster around negatively charged groups (e.g. amino acid R groups) in the active site. This can interfere with the binding of the substrate molecule to the active site.

Question 32

What would be the pH of enzymes which work intracellular lay compared to extracellularly

Answer 32

Enzymes that work intracellularly have an optimum pH that is close to pH 7. Enzymes that work extracellularly may have an optimum pH that is different to pH 7, e.g. enzymes involved in the digestive system.

Question 33

What is a buffer

Answer 33

A buffer is something that resists changes in pH. These donate or accept hydrogen ions to maintain the pH.

Question 34

Explain the effect on increasing substrate concentration at the beginning of the reaction

Answer 34

1) If there is no substrate present, then an enzyme-catalysed reaction cannot take place. 2) As substrate is added, rate of reaction increases as now enzyme-substrate complexes form and so more product forms. 3) Substrate concentration is limiting the reaction, because as it increases, the rate ofreaction increases. The substrate concentration is the limiting factor.

Question 35

Explain the effect on increasing substrate concentration after the Vmax

Answer 35

1) As the concentration of substrate is increased even further, the reaction will reach its maximum rate. 2) Adding more substrate molecules will not increase the rate of reaction because all the active sites are occupied with substrate molecules. 3) Enzyme concentration is now the limiting factor.

Question 36

Sketch a substrate concentration graph

Question 37

Whats is enzyme synthesis

Answer 37

Depending on the cell’s needs, genes for synthesising particular enzymes can be switched on or off.

Question 38

Whats is enzyme degradation

Answer 38

Cells are continuously degrading old enzyme molecules to their component amino acids and synthesising new enzyme molecules.

Question 39

Advantages of rate of synthesis of the enzyme and its rate of degradation

Answer 39

1) The elimination of abnormally shaped proteins that might otherwise accumulate and harm the cell. 2) The regulation of metabolism in the cell by eliminating any unused enzymes.

Question 40

Explain the effect on increasing enzyme concentration at the beginning of the reaction

Answer 40

1) More active sites on the enzyme become available. 2) More successful collisions between the substrate and enzyme occur. 3) More enzyme-substrate complexes can form per unit time, increasing rate of reaction. 4) Enzyme concentration is the limiting factor – as it increases, so does the rate of reaction.

Question 41

Explain the effect on increasing enzyme concentration after the Vmax

Answer 41

1) If substrate concentration is limited/fixed, then all the substrate molecules will be occupying an active site. The reaction is at its maximum rate for this substrate concentration. 2) If the enzyme concentration is increased further, there will be no increase in rate of reaction. 3) The enzyme concentration is no longer the limiting factor, substrate concentration is now the limiting factor.

Question 42

Sketch a enzyme concentration graph

Question 43

Explain why the initial rate of reaction for enzyme-catalyses reaction is the fastest

Answer 43

1) At the beginning of the reaction, there is a greater chance of collision between the enzyme and substrate molecules. 2) As the reaction proceeds, the substrate concentration starts to drop and so does the frequency of successful collisions, slowing the rate of reaction. 3) Thus, the initial reaction rate gives the maximum reaction rate for an enzyme under a particular experimental situation.

Question 44

What are inhibitors

Answer 44

An inhibitor is any substance that slows down or stops the activity of an enzyme. The inhibitor combines with the enzyme in a way that influences how the substrate binds to the enzyme. There are two types of inhibitor: 1) Competitive inhibitors 2) Non-competitive inhibitors Some inhibitors are reversible, others are non-reversible.

Question 45

Why are inhibitors useful

Answer 45

1) In metabolic pathways, it is important that enzyme-catalysed reactions do not happen too fast as this could lead to the build-up of excess products. 2) Living processes are rarely a single reaction, but are complex multi-step pathways that need to be closely regulated to meet the needs of the living organism without wasting resources. 3) The different steps in reaction pathways are controlled by different enzymes. 4) Controlling the activity of enzymes is important to ensure the correct steps in the reaction pathway are taking place and to regulate the rate and quantity of product formation.

Question 46

What are competitive inhibitors

Answer 46

The inhibition that occurs when an inhibitor with the same shape as the substrate combines with the active site, blocking access for the substrate. This type of inhibition is reversed by increasing the concentration of substrate.

Question 47

Explain the process of competitive inhibition

Answer 47

1) The competitive inhibitor fits into the active site and so a substrate molecule cannot enter. 2) The amount of inhibition depends on the relative concentration of substrate and inhibitor molecules. More inhibitor molecules means more inhibitors collide with active sites and so the effect of inhibition is greater. 3) Increasing substrate concentration ’dilutes’ the effect of the inhibitor. 4) The competitive inhibitors compete directly with the substrate for the enzyme’s active site, forming an enzyme-inhibitor complex that is catalytically inactive. 5) Once on the active site, the inhibitor is not changed by the enzyme, but simply prevents the substrate molecule from joining to the active site. 6) Most enzyme inhibition by competitive inhibitors is reversible. If the competitive inhibitor binds irreversibly to the enzyme’s active site, it is called an inactivator.

Question 48

What are non-comepetitive inhibitors

Answer 48

The inhibition that occurs when an inhibitor combines with an allosteric site on the enzyme. The tertiary structure changes and so the active site is no longer able to accept substrate. This type of inhibition is not reversed by increasing the concentration of substrate.

Question 49

Explain the process of non-competitive inhibitors

Answer 49

1) Non-competitive inhibitors do not compete with the substrate molecules for a place on an enzyme’s active site. 2) They attach to another area of the enzyme (known as the allosteric site) away from the active site and disrupt the enzyme’s tertiary structure and change the shape of the active site. 3) This distortion of the active site means that the substrate can no longer bind to the enzyme and so cannot form enzyme- substrate complexes. 4) The maximum rate of reaction is reduced by the presence of non-competitive inhibitors. 5) Adding more substrate will not return the rate of reaction to its uninhibited maximum. The more inhibitor molecules, the greater the reduction in rate of reaction. 6) Enzyme inhibition by non-competitive inhibitors can be either reversible or irreversible. Some remain in the allosteric site, others can leave.

Question 50

Explain reversible inhibition

Answer 50

The inhibition that occurs when an inhibitor combines temporarily with an enzyme. The inhibition is reversed and the enzyme becomes active again when the inhibitor is no longer attached to the enzyme. Reversible inhibitors can be competitive or non-competitive. a) These form weaker hydrogen bonds or weak ionic bonds with the enzyme. b) Their inhibitory effect can be reversed by a change in the environment of the enzyme.

Question 51

Explain non-reversible inhibition

Answer 51

The inhibition that occurs when an inhibitor combines permanently with an enzyme and completely inactivates it. a) These form strong covalent bonds with enzymes. b) The cell must produce more of the enzyme. This can only happen by activating the gene or genes so that the enzymes are transcribed and translated.

Question 52

Explain howare metabolic pathways controlled:

Answer 52

1) In metabolic pathways, it is important that enzyme-catalysed reactions do not happen too fast as this could lead to the build up of excess products. Therefore, end-product inhibition occurs. This is enzyme inhibition that occurs when the product of a reaction acts as an inhibitor to the enzyme that produces it. 2) End-product inhibition is an example of negative feedback. 3) The product of one enzyme-catalysed reaction becomes the substrate for the next enzyme-catalysed reaction in the metabolic pathway. 4) Cells do not need to accumulate too much of the end product, so the product of the last enzyme-catalysed reaction may attach to part of the first enzyme in the pathway and inhibit its use. 5) This would be an example of reversible, non-competitive inhibition. When the concentration of this product within the cell falls, those molecules would detach from the enzyme, allowing the active site to return to its original shape. 6) Multi-enzyme complexes increase the efficiency of metabolic reactions without increasing the substrate concentration.

Question 53

How is the metabolic pathways of respiration controlled

Answer 53

Glucose is broken down in a number of steps: 1) Two phosphate groups added to the glucose molecule. 2) Phosphofructokinase (PFK) catalyses the breakdown of glucose molecule. 3) ATP produced by breakdown of glucose inhibits the PFK enzyme. ATP therefore regulates its own production.

Question 54

Explain how enzyme inhibition protects cells

Answer 54

1) Sometimes, enzymes are secreted as inactive precursors to prevent them from causing cell damage. 2) Part of the precursor molecule is removed by a chemical reaction which activates the enzyme. 3) This is common for protease enzymes to prevent autolysis (self-digestion) of the cell from where they are secreted.

Question 55

What are some examples of inhibitors

Answer 55

a) Poisons include; cyanide, malonate and arsenic. b) Drugs include; penicillin, aspirin, statins and antivirals such as reverse transcriptase inhibitor.

Question 56

What are cofactors

Answer 56

A substance not made from amino acids that is required by an enzyme to function. They may be a permanent part of the molecule (prosthetic group) or a temporary part.

Question 57

What are co-enzymes

Answer 57

A small organic non-protein cofactor. Coenzymes are involved in enzyme catalysed reactions by donating or accepting hydrogen ions or chemical groups such as phosphate groups between different enzyme-catalysed reactions.

Question 59

Describe the differences between a cofactor and a coenzyme

Answer 59

Cofactor: 1) Are inorganic molecules or ions. 2) They work by helping the enzyme and substrate bind together. 3) They don’t directly participate in the reaction so aren’t used up or changed in any way. EXAMPLE: Chloride ions (Cl-) are inorganic cofactors for the enzyme amylase. Amylase will not digest maltose without the presence of the chloride ions. Co-enzyme: 1) Are organic molecules. 2) They participate in the reaction and are changed by it. 3) They often act as carriers, moving chemical groups between different enzymes. 4) They are continually recycled during the process. EXAMPLE: Vitamins are often sources of coenzymes. E.g. NAD acts as a hydrogen carrier (NADH) in reactions of respiration and is derived from vitamin B3.

Question 60

Explain what are prosthetic groups with an example

Answer 60

1) If a cofactor is tightly bound to the enzyme, it is known as a prosthetic group. 2) These prosthetic groups contribute to the 3D shape of the enzyme and are vital to teh active site removing the prosthetic groups could result in the enzyme to become denatured. EXAMPLE: Zinc ions (Zn2+) are a prosthetic group for carbonic anhydrase. This is an enzyme found in red blood cells which catalyses the production of carbonic acid from water andcarbon dioxide). The zinc ions are a permanent part of the enzyme’s active site.

Question 61

Explain what is precursor activation

Answer 61

1) Many enzymes are produced in the inactive form, known as inactive precursor enzymes. 2) This is often the case where enzymes could cause damage to the cells producing them or the tissues where they are released. 3) Precursor enzymes need to undergo a change in shape (tertiary structure), particularly to the active site, to be activated. 4) This can be achieved through the addition of a cofactor. 5) Before the cofactor is added, the precursor enzyme is called an apoenzyme. 6) When the cofactor is added and the enzyme activated, it is called a holoenzyme. Sometimes the change in tertiary structure is brought about by change in environment rather than addition of a cofactor. These types of precursor enzymes are called zymogens or proenzymes.

Question 62

Whats is an example of precursor activation

Answer 62

Inactive pepsinogen is released into the stomach to digest proteins, the acidic pH brings about the transformation into the active enzyme pepsin.

Question 63

Explain a method to look into the effects of enzyme concentration on enzyme activity (PAG 4)

Answer 63

Equipment • 2% Trypsin solution • 2% milk powder solution Distilled water 5 cm syringe 10 cm' measuring cylinder • 9x boiling tubes Stopwatch • Permanent marker pen • Observation sheet 1. Using a serial dilution, the 2% Trypsin solution and distilled water, prepare 1.00, 0.50, 0.25 and 0.13% Trypsin solution 2. Set up five boiling tubes containing 2 cm' 2% milk powder solution solution. Label each with a different concentration of Trypsin solution (i.e. 2.00, 1.00. 0.50, 0.25 and 0.13%). 3. Take the boiling tube labelled 2% Trypsin and position an observation sheet (a piece of paper with written text) behind it. 4. Using a 5 cm' syringe, add 2 cm' 2% Trypsin into the boiling tube and start the stopwatch. 5. The milk powder solution begins opaque. As the reaction progresses, the protein is broken down and the solution becomes clearer. The endpoint of the reaction can be identified when the letters on the sheet become visible. Record this in a suitable table (see below). 6. Repeat steps 3 to 5 for the other Trypsin solution concentrations. 7. Repeat the method a further two times to obtain three repeats for each concentration.

Question 64

Explain the results/ conclusion after looking into the effects of enzyme concentration on enzyme activity (PAGg 4)

Answer 64

Results: As the concentration of Trypsin increases there are a greater number of active sites available. Conclusion: More enzyme-substrate complexes form so the rate of reaction increases. The rate of reaction eventually plateaus as another factor (e.g. substrate concentration) becomes limiting.

Question 65

Explain how we can control an experiment to look into the effects enzyme concentration on enzyme activity (PAG 4)

Answer 65

The variables that are kept constant during the experiment: • Volume of 2% milk powder solution 5 cm' syringe used to measure 2 cm' of 2% milk powder solution • Volume of Trypsin solution 5 cm' syringe used to measure 2 cm' of Trypsin solution • Concentration of milk powder solution 2% milk powder solution used throughout • Temperature and pH Room temperature and pH of the solution remains relatively stable • Same individual used to identify the point at which the writing becomes visible

Question 66

Explain a method to look into the effects of temperature on enzyme activity (PAG 4)

Answer 66

-Suggest a hypothesis -Prepare 4 water baths at different temperatures (e.g. ice bath, 40°C, 60°C, 80°C). Check temperatures with a thermometer and allow them to stabilise. -Add equal volumes and concentrations of hydrogen peroxide (H₂O₂) to 4 boiling tubes. Use a measuring cylinder or 10 cm³ syringe. -Add equal volumes of buffer solution to each tube to keep pH constant. Assembel apparatus by connecting each boiling tube with: -A bung and delivery tube, -An upturned measuring cylinder in a trough of water to collect oxygen. Place each boiling tube in its corresponding water bath for 5 minutes to allow temperature equilibration. Then used a syringe to add the same volume and concentration of catalase to each boiling tube.Immediately seal with the bung and start the timer. -Record the volume of oxygen collected in the measuring cylinder every 30 seconds for 3 minutes. Repeat the experiment at each temperature at least 3 times. Then calculate a mean volume of gas produced for each time point. Calculate Rate of Reaction Use the formula: Rate = total volume of oxygen (cm³) ÷ time (min) for each temperature. Present Data by plotting a graph of rate of reaction (y-axis) against temperature (x-axis).

Question 67

Draw the apparatus used in order to investigate the effect on temperature on enzyme activity (PAG 4)

Question 68

Explain a method to look into the effects of substrate concentration on enzyme activity

Answer 68

1. Set up 5 beakers with varying concentrations of hydrogen peroxide using the serial dilutions technique. Then transfer each solution to a conical flask. 2. Set up the apparatus with a bung attached to an upside-down measuring cylinder in a trough of water by a delivery tube 3. Use a syringe to add the same volume and concentration of catalase to the first conical flask. 4. Quickly put the bung on and start the timer. 5. Record how much oxygen is produced every 30 seconds for 3 minutes. Record this in a results table. 6. Repeat steps 2-5 for each concentration. 7. Repeat at least 2 more times then calculate the mean and standard deviation for each concentration. 8. Calculate the rate of gas production in cm' min' for each concentration of hydrogen peroxide. 9. Draw a graph of rate of reaction against substrate concentration.w

Question 69

Draw the apparatus used in order to look into the effects of substate concentration on enzyme activity (PAG 4)

Question 70

Explain the process of serial dilutions (PAG 4)

Answer 70

By starting with 2.00% of a solution then add 4 cm^3 of this solution to the first boiling tube. Then place 4cm^3 of distilled water in each test tube. After, use a syringe and collect 2cm^3 of the solution from the first tube into the next tube and then mix. Repeat this process but collect from the next test tube until you get percentage of 1.00%,0.50%,0.25% and 0.13%.

Question 71

Explain a method to look into the effects of pH on enzyme activity (PAG 4)

Answer 71

Apparatus: • Test tubes • Buffer solutions at different pH levels • Amylase solution • lodine solution • Starch solution • Pipettes • Spotting tile • Timer • Gloves • Goggles Method: • Wear goggles and gloves • Enzymes have the potential to cause allergic reactions if they come into direct contact with skin • Place single drops of iodine solution in rows on the tile • lodine solution is orange-brown • Label a test tube with the pH to be tested • Use the syringe to place 2cm3 of amylase in the test tube • Equal volume and concentration of enzyme should be used so these variables are controlled and the effect of changing pH can be measured • Add lcm' of buffer solution to the test tube using a syringe • Use another test tube to add 2cm' of starch solution to the amylase and buffer solution, start the stopwatch whilst mixing using a pipette • Equal volume and concentration of the substrate (starch) should be used so these variables are controlled and the effect of changing pH can be measured • Mixing enables the enzymes and substrate to be equally mixed • After 10 seconds, use a pipette to place one drop of the mixture on the first drop of iodine, which should turn blue-black • This test indicates whether starch is still present • Wait another 10 seconds and place another drop of the mixture on the second drop of iodine Repeat every 10 seconds until iodine solution remains orange-brown • When the solution remains orange-brown it means amylase has broken down all of the starch so nothing is left to react with the iodine • Repeat experiment at different pH values • The less time the iodine solution takes to remain orange-brown, the quicker all the starch has been digested and so the better the enzyme works at that pH

Question 73

Explain the limitations to look into the effects of pH on enzyme activity (PAG 4)

Answer 73

Limitations: • The above method can be adapted to control temperature by using a water bath at 35°C • All solutions that need to be used (starch, amylase, pH buffers) should be placed in a water bath and allowed to reach the temperature (using a thermometer to check) before being used • A colorimeter can be used to measure the progress of the reaction more accurately; with a solution containing starch being darker and glucose lighter (as a result of the colour-change of iodine) - this will affect the absorbance or transmission of light in a colorimeter