B4: Enzymes
Enzymes with very low optimum temperatures tend to have quite flexible structures. Using your knowledge of collision theory, explain why this flexibility is necessary.
(at low temperatures) Kinetic energy is low (1); substrates / enzymes, move slowly (1); (so) fewer collisions (1); collisions have less energy (1); increased flexibility of active site (1); increases chances of successful collision (1)
PQ2ii: Amylase catalyses the hydrolysis of starch to maltose in the mouth. Amylase stops working as the food passes into the stomach. Maltase then catalyses the hydrolysis of maltose into glucose in the small intestine. (i) State which group of biological molecules amylase and maltase belong to. (ii) Describe the meaning of the term hydrolysis. (2 marks) (iii) Explain why maltase, not amylase, hydrolyses maltose completing the digestion of starch. (iv) Explain why amylase stops working in the stomach.
(i) enzymes. (ii) break bond; involving the addition of water; (iii) enzymes specific; amylose is hydrolysed to maltose; active site of maltase is complementary to maltose; (iv) maltose hydrolysed / digested, by enzymes; enzymes are proteins; stomach contents contain proteases; proteases hydrolyse proteins;
PQ3iv: (i) Explain why end product inhibitors have to be both reversible and competitive. (ii) Explain why end product enzyme inhibition is essential for the control of cellular activity.
(i) idea of control mechanism; need to be reversible so more product can be made, when needed; with competitive inhibitor, more substrate would overcome inhibition; (leading to) excess product (ii) prevent build-up of, excess / unnecessary products; maybe toxic; prevent waste of resources / energy; ensure sufficient levels of required products;
PQ5: Figure 4 shows the results that the student obtained from a practical procedure in which the rate of formation of maltose was measured in the presence and absence of chloride ions. (i) Describe the effect of chloride ions on the rate of reaction. (Ii) State three variables that need to be controlled in this practical procedure in order to produce valid results.
(i) increases / greater / faster ; reaction completed in / plateaus after / concentration is 100% after, 3.5 minutes ; figures with units to support mp 1 ; (ii) temperature ; pH ; enzyme / amylase / chloride, concentration ; substrate / starch / amylose, concentration ; constant / regular, stirring ; (fixed) volume of solution (removed each time for sampling) ;
PQ2iii: The acid stomach contents are neutralised by alkaline pancreatic juice in the small intestine. (i) Suggest why this is necessary to ensure digestion of maltose. (ii) Explain what is meant by the term Vmax with respect to enzymes.
(i) stomach contents are acidic; optimum pH of, enzymes / named, is at higher pH; (pancreatic) enzymes not denatured; (ii) reaction is at maximum rate; all active sites occupied; enzyme activity is maximum;
How is starch digested?
-Salivary amylase in the mouth and pancreatic amylase in the small intestine breaks the starch into maltose -Maltase on the lining of the small intestine breaks down the maltose into glucose Glucose is small enough to be absorbed by the cells lining in the digestive system and subsequently absorbed in the bloodstream.
What are the two hypotheses of enzyme and substrate interactions?
1) Lock and Key Model. 2) Induced Fit Model.
PQ2: Describe what is happening at the points labelled on the graph below. You should use the terms active site, substrate and enzyme substrate complex in your answer.
A / rate of reaction increasing active sites available; increasing number of collisions between active sites and substrates; more enzyme substrate complexes forming; B / reaction rate constant all active sites occupied by substrate; idea of substrate queuing up for active sites; Vmax;
What are statins?
A competitive inhibitor used in the synthesis of cholesterol which help to lower blood cholesterol concentration e.g. prevent things such as heart disease,
Explain why a non-competitive inhibtor does not need to have a similar shape to a substrate molecule.
A non-competitive inhibitor binds to an enzyme away from the active site (1) at an allosteric site (1), which has a different shape than the active site (1).
What are cofactors and coenzymes?
A non-protein component which aid the enzyme in performing its function as a biological catalyst. They may help transfer atoms or groups of atoms in a multi-step pathway or they may form part of the active site. If the component is organic it is called a coenzyme.
What is aspirin?
A permanent competitive inhibitor that inhibits the active site of COX enzyme, preventing the synthesis of prostaglandins and thromboxane, the chemicals responsible for producing pain and fever.
State the type of biological molecule that forms enzymes.
A protein.
3a/b/c/d.) Some mice have diabetes. The diabetes causes the blood glucose concentration to become very high after a meal. Scientists investigated the use of an inhibitor of amylase to treat diabetes. The scientists took 30 mice with diabetes and divided them into two groups, A and B. Group A was given yoghurt without the inhibitor of amylase each day. Group B was given yoghurt with the inhibitor of amylase each day. Apart from the yoghurt, all of the mice were given the same food each day. The scientists measured the blood glucose concentration of each mouse, 1 hour after it had eaten. This was done on days 1, 10 and 20 after the investigation started. The following figure shows the scientists' results. A.) Explain the use of a control. B.) Apart from the yoghurt, it was important that all of the mice were given the same food each day. Give two reasons why it was important that all of the mice were given the same food each day. C.) The scientists' hypothesis was that adding the inhibitor of amylase to the food would lead to a lower blood glucose concentration. Use your knowledge of digestion to suggest how the addition of the inhibitor could lead to a lower blood glucose concentration. D.) Give one reason why these results may not support the use of the inhibitor of amylase to treat diabetes in mice.
A.) to show the effect of the inhibitor, to show the effect of the yoghurt (on its own it does not affect blood glucose) B.) Food is a factor that affects blood glucose / different foods contain different amounts of glucose to keep starch/fibre intake similar. C.) fewer enzyme - substrate complexes formed. Less starch digested to maltose. Less glucose from maltose. Less absorption of glucose from gut. D.) need larger sample of mice so might not be representative.
4a.) An enzyme catalyses only one reaction. Explain why.
Active site is specific to a specific substrate
How do you activate precursor enzymes?
Add a cofactor. Precursor enzymes need to undergo a change in tertiary structure, particularly to the active site, to be activated. Before the enzyme is activated it is called an aplenty egg. After the cofactor is added and the enzyme is activated, it is called a holoenzyme.
4b.) Gout is a disease caused by the build-up of uric acid crystals in joints. Uric acid is produced from xanthine in a reaction catalysed by the enzyme xanthine oxidase. Xanthine —————————> Uric acid Xanthine oxidase Allopurinol is a drug used to treat gout..The diagram shows the structures of xanthine and allopurinol. Use this information to suggest how allopurinol can be used to treat gout.
Allopurinol has a similar shape to xanthine. Allopurinol enters active site/ is a competitive inhibitor Less xanthine binds, fewer enzyme-substrate complexes, fewer Uris acid crystals formed, less uric acid formed
Name the monomers that form a protein.
Amino acids.
PQ3: State the type of monomers that make up proteins.
Amino acids.
What is an inactive precursor enzyme?
An enzyme in an inactive form, particularly enzymes that can cause damage within cells producing them or to tissues where they are released or enzymes whose action needs to be controlled and only activated under certain conditions.
What are extracellular enzymes?
An enzyme secreted by a cell and functions outside of that cell.
What are intracellular enzymes?
An enzyme that functions within the cell it was produced.
What type of chemical reactions are required for growth?
Anabolic (catalysed by enzymes).
PQ3iii: Identify which curve on the graph below demonstrates competitive inhibition giving the reasons for your choice.
B -Reaction rate of B eventually equals A
Bacteria that colonise hydrothermal vents, where temperatures are very high, have enzymes with very high optimum temperatures. Suggest why these bacteria are unlikely to cause infections in humans.
Bacterial enzymes have high optimum temperatures, human body temperature is lower (1). Enzymes will have low activity (1) and bacteria will not thrive (1).
What are enzymes?
Biological catalysts.
Explain the energy changes resulting in the transition state model.
Bonds are broken (1); in substrate (1); energy required (1); energy (of system (increases) (1)
Explain how the following terms are relevant to each of the models: complementary, flexibility, R group interactions, bond strain.
Both models substrate interacts with R groups in active site (binds) (1); (leading to) bond strain in substrate molecule (1); Lock and key substrate is complementary to active site (of enzyme) (1); Induced fit active site is flexible (1); (active site) changes shape as substrate binds, closer fit between active site and substrate.
Explain how catabolism and anabolism are related to metabolism.
Catabolism is breaking down of molecules (1); anabolism is building of molecules (1); reactions involve breaking down and building of molecules (1); idea of metabolism is sum of all reactions (1).
What are catabolic reactions?
Chemical reactions that break large molecules apart.
Explain, using an appropriate example of each, how prosthetic groups are different from coenzymes.
Coenzymes bind loosely to enzymes (1); e.g. NAD (1); prosthetic groups are a permanent feature of / bind tightly to, proteins / enzymes (1); e.g. iron ion in haemoglobin (1).
2d.) Explain the difference in the rates of reaction at 60 °C and 37 °C between 20 and 40 minutes.
Different times. Higher temperature causes desaturation of all enzymes Reaction stops sooner because active site shape has changed Different concentrations of product (at 60) Substrate still available But not converted to product.
Give an example of a multi-cellular organism using extracellular enzymes.
Digestion within humans using amylase and trypsin. The products are transported in the bloodstream around the body.
End-product inhibition is likely to be competitive rather than non-competitive. Suggest reasons for this, and give an example of end-product inhibition.
End-product inhibition regulates rate of reaction (1); concentrations of substrate and product determine reaction rate (1); (so must be) competitive (1); substrate concentration has no effect in non- competitive inhibition (1); e.g. ATP and PFK in respiration (1).
Ethylene glycol present in anti-freeze is poisonous when ingested. Ethylene glycol is oxidised using the enzymes used to oxidise ethanol (alcohol). The products made during the breakdown of ethylene glycol, rather than ethylene glycol itself, are responsible for the toxic effects. Ethylene glycol is able to leave the body unchanged in urine. Suggest why ethanol is often used in emergency departments as an antidote to antifreeze poisoning.
Ethanol has similar shape to ethylene glycol (1); (ethanol) binds to active site of enzyme which breaks down ethylene glycol (1); competitive inhibition (1); less ethylene glycol broken down (1); more (ethylene glycol) leaves body unchanged (1); fewer toxic effects (1).
6b.) The binding of the functional form of TK to its substrate leads to cell division. Chronic myeloid leukaemia is a cancer caused by a faulty form of TK. Cancer involves uncontrolled cell division. Figure 2 shows the faulty form of TK. Suggest how faulty TK leads to chronic myeloid leukaemia.
Faulty TK has functional active site without phosphate So faulty TK functional all the time (not controlled by phosphate)
What is the function of trypsin?
Found in the pancreas, it is secreted into the small intestine where it breaks down proteins. The amino acids produced by the action of protease are absorbed by the cells lining in the digestive system and then absorbed into the bloodstream.
Discuss why the models of enzyme action have changed over time.
Idea of improved technology (1); idea of continually investigated (1); more evidence (1); more accurate representation (1).
What is the effect of non-competitive inhibitors on rate of reaction?
Increasing the concentration of enzyme or substrate will non overcome the effect of a non-competitive inhibitor. Increasing the concentration of inhibitor, however, will decrease the rate of reaction further as more active sites become unavailable.
What does a plateaued line on a concentration vs rate of reaction graph mean?
Increasing the concentration of the substrate increases the numbers of substrate molecules that can form enzyme-substrate (ES) complexes at any one time. This increases the initial rate of reaction but when all the enzyme molecules are engaged in ES complexes the rate cannot increase any further. The rate will then plateau because the enzyme is said to be saturated.
Explain why increasing the concentration of substrate will never produce the Vmax of a reaction after the addition of a non-competitive inhibitor.
Inhibitor will always be present (1); some enzymes always inhibited (1).
What are inhibitors?
Inhibitors are compounds (many of which are drugs) that inhibit the activity of the enzyme.
1c.) Describe and explain the differences between the two curves.
Initial rate of reaction faster at 37 degrees Celsius Because more kinetic energy. So more enzyme-substrate collisions/ complexes Graph reaches plateau at 37 degrees Because all substrate is used up
What are the effects of competitive inhibitors?
It reduces the rate of reaction for a given concentration of substrate, but it does not change the Vmax (maximum rate) of the enzyme it inhibits. e.g. if substrate concentration is increased enough there will be a larger proportion of substrate to inhibitor so Vmax can still be reached.
Explain the meaning of the term activation energy.
Minimum energy required (1); to start reaction (1)
What are competitive inhibitors?
Molecules that bind to the active site of an enzyme and inhibit the ability of the substrate to bind.
2c.) Explain the difference in the initial rate of reaction between enzyme activity at 60 degrees Celsius and 37 degrees Celsius.
More kinetic energy and more enzyme-substrate complexes formed.
IMantib is a frug used to treat chronic myeloid leukaemia.
Non competitive inhibitor binds to almost Eric site Causes TK to be in non-functional form ' active site not deformed ' wrong shape / ES complex not formed So uncontrolled cell division stopped
What are non-competitive inhibitors?
Non-competitive inhibitors bind at a location other than the active site, which results in a change of shape in the enzyme so that the enzyme cannot bind to the substrate.
6a.) The enzyme tyrosine kinase (TK) is found in human cells. TK can exist in a non- functional and a functional form. The functional form of TK is only produced when a phosphate group is added to TK. Addition of a phosphate group to the non-functional form of TK leads to production of the functional form of TK. Explain how.
Phosphate changes shape of TK / changes shape of enzyme / changes active site Active site forms /. Becomes the right shape / can bind to substrate / complementary to substrate
Using blood clotting as your example, explain the different ways in which enzymes can be activated.
Presence of cofactor (1); e.g., vitamin K and Factor X (1); change in tertiary structure / described (1); e.g. (activated) factor X catalyses the breaking of bonds in prothrombin (1); forming thrombin (1); thrombin catalyses the conversion of fibrinogen to fibrin (1).
Explain the term "denatured" with reference to enzymes.
R-group interactions are disrupted (1); change in tertiary structure (1); change in 3D shape of active site preventing binding with substrate (1).
How is the production of ATP an example of end-product inhibition?
Respiration is a metabolic pathway resulting in the production of ATP. Glucose is broken down in a number of steps: The first step involves the addition of two phosphate groups to the glucose molecule. The addition of the second phosphate group, which results in the initial breakdown of the glucose molecule, is catalysed by the enzyme phosphofructokinase (PFK). This enzyme is competitively inhibited by ATP. ATP therefore regulates its own production. • When the levels of ATP are high, more ATP binds to the allosteric site on PFK, preventing the addition of the second phosphate group to glucose. Glucose is not broken down and ATP is not produced at the same rate. • As ATP is used up, less binds to PFK and the enzyme is able to catalyse the addition of a second phosphate group to glucose. Respiration resumes, leading to the production of more ATP.
What are prosthetic groups?
Similar to cofactors, but they are tightly bound to the enzyme, rather than being separate molecules or atoms and form a permanent feature of the protein. e.g. zinc ions form an important part of the structure of carbonic a hydrate, an enzyme necessary for the metabolism of CO2.
"The lock and key model and induced fit model". Explain what is meant by the term 'model' in the sentence above.
Simple / easy to understand (1); representation (1).
Describe how the structure if proteins determines enzyme activity.
Specific, 3D shape / tertiary structure (1); (formation of) active site (1); binds to substrate(s) (1); catalyses reaction (1)
PQ1: Compounds containing some metal ions such as lead, mercury, copper or silver are poisonous. This is because ions of these metals are non-competitive inhibitors of several enzymes. Other metal ions are required for enzymes to function. Magnesium s, for example, are required by DNA polymerase. Which of the following statements correctly describes the nature and function of magnesium ions? Statement 1: minerals and coenzymes Statement 2: vitamins and cofactors Statement 3: minerals and cofactors Statement 4: vitamins and coenzymes
Statement 3
What factors affect enzyme activity?
Temperature, substrate/enzyme concentration and pH.
What is end-product inhibition?
Term used for enzyme inhibition that occurs when the product of a reaction acts as an inhibitor to the enzyme that produces it. This acts as a negative-feedback control mechanism for the reaction. Excess products are mot made and resources are not wasted, it is an example of non-competitive reversible inhibition.
What is the induced fit hypothesis?
The active site will change shape to fit the substrate. The initial interaction between the enzyme and substrate is relatively weak, but these weak interactions rapidly induce changes in the enzyme's tertiary structure that strengthen binding, putting strain on the substrate - this can weaken the substrate and lower the activation energy.
What is an enzyme-substrate complex?
The binding and interactions of a substrate to the active site of an enzyme.
How do hydrogen ions affect enzymes?
The hydrogen ions interact with the charged R-groups on the amino acids, changing the concentration of H+ ions therefore affects the degree of this interaction, The more acidic the solution, the more H+ ion present, the less the R-groups are able to interact with each other. This leads to bonds breaking and the shape of the enzyme changing.
What is the lock and key hypothesis?
The idea that the enzyme active site has a fixed shape and only the right substrate will fit into the active site. An enzyme-substrate complex is formed when the substrate is bound to the active site. The substrate then reacts and products form within the enzyme-product complex.
What is the effect of an increased number of substrate particles on rate of reaction?
The increased number of substrate particles leads to a higher collision rate with the active sites of enzymes and the formation of more enzyme-substrate complexes. So the rate of reaction increases>
What is Vmax of an enzyme?
The maximum rate of reaction or the rate of reaction under ideal circumstances.
What is activation energy?
The minimum required amount of energy for a reaction to start.
What is the link between pH and hydrogen ions present in the solution?
The more acidic the solution the more H+ ions in solution.
Why are competitive inhibitors called competitive inhibitors?
The non-substrate molecule that binds to the active site is a type of inhibitor. Substrate and inhibitor molecules present in a solution will compete with each other to bind to the active sites of the enzymes catalysing the reaction. This will reduce the number of substrate molecules binding to active sites in a given time and slows down the rate of reaction. For this reason such inhibitors are called competitive inhibitors and the degree of inhibition will depend on the relative concentrations of substrate, inhibitor, and enzyme.
What is the temperature coefficient of a reaction?
The temperature coefficient, Q10, of a reaction I'd a measure of how much the rate of a reaction increases with a 10 degree Celsius rise in temperature. For enzymes the temperature coefficient is usually 2 (rate of reaction doubles with a 10 degree Celsius temperature increase).
What happens to enzyme activity as you increase the temperature until optimum temperature?
The temperature of the environment increases, there is more kinetic energy within the enzymes, they move faster, there are more frequent collisions, there are more frequent successful collisions, this leads to an increased rate of reaction.
Why do enzymes denature?
The tertiary structure bonds begin to break within the enzyme causing the active site to change shape. The enzyme is no longer complementary to the substrate and can no longer function as a catalyst.
How are coenzymes obtained?
They are derived from vitamins, a class of organic molecule found in the diet. e.g. vitamin B3 is used to synthesise NAD (nicotinamide adenine dinucleotide), a coenzyme responsible for the transfer of hydrogen atoms between molecules involved in respiration.
What are metabolic pathways?
They are sequences of enzymatically catalyzed chemical reactions in a cell.
What are organphosphates?
They are used as insecticides and herbicides as a non-competitive inhibitor. They irreversibly inhibit the enzyme acetyl cholinesterase, an enzyme necessary for nerve impulse transmission. This can lead to muscle cramps, paralysis, and even death if ingested.
What are proton pump inhibitors (PPIs)?
They are used to treat long term indigestion as a non-competitive inhibitor. They irreversibly block an enzyme system responsible for secreting hydrogen ions into the stomach. This makes PPIs very effective in reducing the production of excess acid, which if left untreated, can lead to stomach ulcers.
What adaptations do enzymes have that need to function as cold temperatures?
They have flexible structure (especially at the active site) making them less stable.
What adaptations do enzymes have that need to function as hot temperatures?
They have increased amount of hydrogen and sulphur bonds within their tertiary structure causing a higher stability. The shapes of the enzyme is more resistant to change in temperature.
Describe two ways in which cofactors are necessary for the catalytic role of some enzymes.
Transfer, atoms / groups, between reactions (1); form part of active site (1).
How are inorganic cofactors obtained?
Via the diet as minerals, including calcium, iron, chloride, and zinc ions e.g. the enzyme amylase, which catalyses the breakdown of starch, contains a chloride ion that is necessary for the formation of a correctly shaped active site.
What is renaturation?
When a enzyme returns to its original shape.
What are zymogens/proenzymes?
When another enzyme causes a change in the tertiary structure of the precursor enzyme e.g. protease which cleaves certain bonds within a molecule. In addition, a zymogen/proenzyme could be formed when a change in pH or temperature occurs which results in a change of tertiary structure. e.g. when inactive pepsinogen is released into the stomach to digest proteins, the acid pH activates the enzyme. This adaptation protects the body tissues against the digestive action of pepsin.
What is an enzyme-product complex?
Where the products of the substrate are formed.
Give an example of a single-cell organism using extracellular enzymes.
Yeast releases enzymes into their immediate environment. The enzymes break down larger (polymer) molecules, such as proteins, and the products such as amino acids are absorbed by the cell.
PQ3ii: Malonate inhibits cellular respiration. Hans Krebs added malonate to minced pigeon muscle tissue when trying to work out the different steps in Krebs cycle, a series of reactions that occur in respiration. In step 6 of Krebs cycle, succinate dehydrogenase catalyzes the oxidation of succinate to fumarate. Explain why malonate inhibits this reaction.
malonate is similar shape to substrate / succinate; part of / group on, malonate the same as on succinate; malonate, fits into / is complementary to, active site of enzyme; prevents substrate binding; competitive inhibition;
2a.) A technician investigated the effect of temperature on the rate of an enzyme-controlled reaction. At each temperature, he started the reaction using the same concentration of substrate. Give two other factors the technician would have controlled.
pH, volume of substrate and concentration of enzyme
1a.) A technician investigate the effect of temperature on the rate of an enzyme-controlled reaction. Give one other factor the technician would have controlled.
pH/concentration of substrate solution/of enzyme
PQ4: Amylase is an enzyme that hydrolyses amylose to maltose. Maltose, like glucose, is a reducing sugar. A student investigated the action of amylase on amylose. She mixed amylase with amylose and placed the mixture in a water bath. Describe how she could measure the change in concentration of maltose (reducing sugar) as the reaction proceeds. In you answer, you should ensure that the steps in the procedure are sequenced correctly.
take samples at a range of times / AW ; same volumes (of solutions) added / removed (each time) ; heat with, Benedict's (solution) / CuSO4 and NaOH ; (use of ) excess Benedict's ; changes to, green / yellow / orange / brown / (brick) red ; remove precipitate / obtain filtrate ; colorimeter ; calibrate / zero, using, a blank / water / (unreacted) Benedict's ; use (red / orange) filter ; reading of, transmission / absorbance OR mass of precipitate ; more transmission / less absorbance, of filtrate, OR greater mass ppt, = more maltose present ; ora using, standard / known, concentrations (of maltose) ; (obtain) calibration curve ; plot, transmission / absorbance / mass of ppt, against (reducing sugar) concentration ; use graph to read off concentration of maltose / AW ;
How do competitive inhibitors work?
• A molecule or part of a molecule that has a similar shape to the substrate of an enzyme can fit into the active site of the enzyme. • This blocks the substrate from entering the active site, preventing the enzyme from catalysing the reaction. • The enzyme cannot carry out its function and is said to be inhibited. Most competitive inhibitors only bind temporarily to the active site of the enzyme so their effect is reversible - however their are exceptions e.g. aspirin.
How do non-competitive inhibitors work?
• The inhibitor binds to the enzyme at a location other than the active site. This alternative site is called an allosteric site. • The binding of the inhibitor causes the tertiary structure of the enzyme to change, meaning the active site changes shape. • This results in the active site no longer having a complementary shape to the substrate so it is unable to bind to the enzyme. • The enzyme cannot carry out its function and it is said to be inhibited.
