1. Enzymes
2.5, 8.1

2. Enzymes 2.5 Understanding (Statement objectives)
Define enzyme and active site (to which specific substrates bind).
State enzyme catalysis involves molecular motion and the collision of substrates with the active site.
State temperature, pH and substrate concentration affect the rate of activity of enzymes.
State that enzymes can be denatured.
State that immobilized enzymes are widely used in industry.
Applications
Explain the use of lactase in the production of lactose-free milk.
Describe methods of production of lactose-free milk and its advantages.
Lactase can be immobilized in alginate beads and experiments can then be carried out in which the lactose in milk is hydrolysed.
Nature of science
Experimental design—State that accurate, quantitative measurements in enzyme experiments require replicates to ensure reliability.
Draw and label graphs to show the expected effects of temperature, pH and substrate concentration on the activity of enzymes.
Explain patterns and trends in these graphs.
Skills
(Design of experiments to test) Explain the effects of temperature, pH and substrate concentration on the activity of enzymes.
Experimental investigation of a factor affecting enzyme activity.
Explain that enzymes lower the activation energy of the chemical reactions that they catalyze.
Explain the difference between competitive and noncompetitive inhibition with reference to one example of each.
Explain the control of metabolic pathways by end-product inhibition, including the role of allosteric sites.

3. Enzymes 8.1 Understanding
Metabolic pathways consist of chains and cycles of enzyme-catalysed reactions.
Enzymes lower the activation energy of the chemical reactions that they catalyse.
Enzyme inhibitors can be competitive or non-competitive.
Enzyme inhibition should be studied using one specific example for competitive and non-competitive inhibition.
Metabolic pathways can be controlled by end-product inhibition.
Applications
Explain the end-product inhibition of the pathway that converts threonine to isoleucine.
Explain the use of databases to identify potential new anti-malarial drugs.
Nature of science
Developments in scientific research follow improvements in computing—Describe developments in bioinformatics, such as the interrogation of databases, have facilitated research into metabolic pathways.
Skills
Calculate and plot rates of reaction from raw experimental results.
Distinguish different types of inhibition from graphs at specified substrate concentration

4. Enzymes

5. Enzymes control cell metabolism
Enzymes are:
globular proteins
biological catalysts that speed up reactions
they are not used up in a reaction
Lower activation
energy of a reaction

6. Enzyme- Substrate Specificity
Enzymes are found in all living cells
Living organisms produce many different enzymes
Enzymes only catalyse one biochemical reaction- this is called enzyme- substrate specificity
The shape and chemical properties of the active site and substrate match each other
Substrate can bind, but not other substances

9. Enzyme-substrate complex
Once an enzyme binds with its substrate, it is called an enzyme-substrate complex
In order to bind, the substrate must collide with the enzyme
Collision theory

10. Everything you need to know
Something more interactive

11. Manipulating Enzymes
Enzymes are important resources for many industries
For example:
Lactase is used in milk to create lactose-free milk (more on this later)
Restriction enzymes used in plasmids to create recombinant DNA
Taq polymerase used in PCR reactions

12. Common uses of enzymes in industry include:
Detergents contain proteases and lipases to help breakdown protein and fat stain
In the textiles industry enzymes help in the processing of fibres, e.g. polishing cloth to make it appear more shiny
Enzymes are used to breakdown the starch in grains into biofuels that can be combusted

13. Common uses of enzymes in industry include:
Enzymes are widely used in the food industry, e.g.
fruit juice, pectin to increase the juice yield from fruit
Fructose is used as a sweetener, it is converted from glucose by isomerase
Rennin is used to help in cheese production

14. Enzymes used in industry are usually immobilized
Enzymes used in industry are usually immobilized. They are attached to a material so that their movement is restricted. Common ways of doing this are:
Aggregations of enzymes bonded together
Attached to surfaces, e.g. glass
Entrapped in gels, e.g. alginate gel beads

15. Advantages of enzyme immobilization:
Concentration of substrate can be increased as the enzyme is not dissolved – this increases the rate of reaction
Recycled enzymes can be used many times, immobilized enzymes are easy to separate from the reaction mixture, resulting in a cost saving. o
Separation of the products is straight forward (this also means that the the reaction can stopped at the correct time).
Stability of the enzyme to changes in temperature and pH is increased reducing the rate of degradation, again resulting in a cost saving.

16. Production of Lactose-free milk
Lactase obtained from commonly from yeast (bacteria is an alternative)
Lactase is bound to the surface of alginate beads
Milk is passed (repeatedly) over the beads
The lactose is broken down into glucose and galactose
The immobilized enzyme remains to be used again and does not affect the quality of the lactose free milk

17. Other uses of lactose free milk:
As a means to increase the sweetness of milk (glucose and galactose are sweeter in flavour), thus negating the need for artificial sweeteners
As a way of reducing the crystallisation of ice-creams (glucose and galactose are more soluble than lactose)
As a means of shortening the production time for yogurts or cheese (bacteria ferment glucose and galactose more readily than lactose)

18. HIGHER LEVEL

19. More on activation energy

20. How do enzymes lower the activation energy of a reaction?
The substrate binds to the enzymes’ active site and the active site is altered to reach the transition state.
Due to the binding the bonds in the substrate molecule are stressed/become less stable.
The binding lowers the overall energy level of the transition state.
The activation energy of the reaction is therefore reduced.

21. Anabolic Reactions
small molecules are used as building blocks to create larger ones
“Biosynthesis”
Two substrates enter a enzyme and chemical bonds are formed to create one macromolecule
Require energy to form these bonds
endothermic

22. Catabolic Reactions Catabolic reactions
Used to break down larger molecules into smaller subunits
Enzyme is used to break one substrate into two
Thus chemical bonds are broken, releasing energy
exothermic

23. Examples?

24. However...
Body rarely achieves the required product after just one reaction
Thus, metabolic pathways consists of chains and cycles of enzyme-catalysed reactions

25. Metabolic pathways

26. End-Product Inhibition
End-product inhibition prevents the cell from wasting chemical resources and energy by making more of a product than it needs
Metabolic reactions occur in an assembly line fashion to reach a specific end product

27. End-Product Inhibition
Once there is enough end-product, the whole metabolic pathway shuts down
The end-product binds to the very first enzyme allosterically
As the end-product is used up, the enzyme is reactivated
In high concentrations, the end-product inhibits.
In low concentrations, no inhibition.

29. Glycolysis, a part of respiration, is an example of a metabolic chain
Examples
Glycolysis, a part of respiration, is an example of a metabolic chain
The Calvin cycle, a part of photosynthesis, is an example of a metabolic cycle

30. Enzyme inhibitors can be competitive or non-competitive.

31. Enzyme inhibitors can be competitive or non-competitive.

32. Enzyme inhibitors can be competitive or non-competitive.

33. Enzyme inhibitors can be competitive or non-competitive.

34. Enzyme inhibitors can be competitive or non-competitive.

35. Features of competitive inhibitors
Distinguishing different types of inhibition from graphs at specified substrate concentration.
Features of competitive inhibitors
Rate of reaction is reduced
When the concentration of substrate begins to exceed the amount of inhibitor, the maximum rate of the uninhibited enzyme can be achieved. However, it takes a much higher concentration of substrate to achieve this maximum rate.

36. Features of non-competitive inhibitors
Distinguishing different types of inhibition from graphs at specified substrate concentration.
Features of non-competitive inhibitors
Rate of reaction is reduced
The binding of the non-competitive inhibitor prevents some of the enzymes from being able to react regardless of substrate concentration.
Those enzymes that do not bind inhibitors follow the same pattern as the normal enzyme.
It takes approximately the same concentration of enzyme to reach the maximum rate, but the maximum rate is lower than the uninhibited enzyme.

37. Enzyme inhibitors can be competitive or non-competitive.

38. Isoleucine is an essential amino acid*
End-product inhibition of the pathway that converts threonine to isoleucine.
Isoleucine is an essential amino acid*
Bacteria synthesize isoleucine from threonine in a series of five enzyme-catalysed steps
As the concentration of isoleucine increases, some of it binds to the allosteric site of threonine deaminase
Isoleucine acts as a non-competitive inhibitor to threonine deaminase
The pathway is then turned off, regulating isoleucine production.
If the concentration of isoleucine later falls (as a result of its use) then the allosteric sites of threonine deaminase are emptied and the enzymes recommences the conversion of threonine to isoleucine takes place.
*Essential amino acids cannot be made by the body, therefore they must come from food.

39. Denaturation
Enzymes are proteins, therefore they get denatured as well
Denaturation effects both the primary and tertiary structures of an enzyme
this causes a loss of shape and change in activation site
Once an enzyme has lost its shape, it can no longer bind with its substrate

40. Temperature Denaturation
Rise in heat energy causes atoms of the enzyme to get excited
They begin to vibrate, causing the weaker bonds to break
Once the bonds break, the activation site is altered, disallowing the substrate from bonding

41. pH Denaturation
H+ will change the electrostatic attraction between two amino acids
When pH is altered, H+ concentrations will either increase or decrease, attaching themselves to the enzyme
This causes new bonds to form or break
Once bonds are altered, the shape of the enzyme is also altered

42. Use of databases to identify potential new anti-malarial drugs.
Bioinformatics is an approach whereby multiple research groups can add information to a database enabling other groups to query the database.
Bioinformatics has facilitated research into metabolic pathways is referred to as chemogenomics.
Sometimes when a chemical binds to a target site, it can significantly alter metabolic activity.
Massive libraries of chemicals are tested individually on a range of related organisms.
For each organism a range of target sites are identified.
A range of chemicals which are known to work on those sites are tested.

44. Use of databases to identify potential new anti-malarial drugs.
Malaria is a disease caused by the pathogen Plasmodium falciparum.
This protozoan uses mosquitoes as a host as well as humans and hence can be passed on by mosquito bites
Increasing drug resistance to anti-malarial drugs has lead to the use of bioinformatics and chemogenomics to try and identify new drugs.
In one study, approx. 300,000 chemicals were screened against a chloroquine-sensitive 3D7 strain and the chloroquine-resistant K1 strain of P. falciparum.
Other related and unrelated organisms, including human cell lines, were also screened.
(19) new chemicals that inhibit the enzymes normally targeted by anti-malarial drugs were identified
Additionally (15) chemicals that bind to malarial proteins were identified – this can help in the location of P. falciparum
These results indicate possible new directions for drug research.

45. Review...

51. Environmental effects on rate activity of enymes
Three things affect enzyme activity rate
Temperature pH
Substrate Concentration
You need to be able to draw and clearly explain their effects!
Simulation

52. Temperature At low temperatures, very little enzyme activity
This is because not enough heat energy present to initiate collision and bond formation
As temperature increases, enzyme activity also increases
This occurs until temperatures increases to a specific point
The enzyme then denatures
Thus, enzymes have a narrow temperature in which they function

53. pH enzymes have an optimal pH
Range deviating from optimal is very narrow
Too acidic or too basic and the enzyme will begin to denature
However, pH within the body varies greatly, so enzymes adapt to suit the pH they operate in
Pepsin is an enzyme in the stomach to break down food. Why is it’s optimal pH so low?

54. Substrate Concentration
Enzyme activity increases as substrate concentration increase
This is until substrate concentration surpasses enzyme concentration
Then, enzyme activity plateaus
Why?

55. What is the effect of substrate concentration?
Design of experiments to test the effect of temperature, pH and substrate concentration on the activity of enzymes. Experimental investigation of a factor affecting enzyme activity. (Practical 3)
Catalase is one of the most widespread enzymes. It catalyses the conversion of hydrogen peroxide, a toxic by-product of metabolism, into water and oxygen.
H2O2
Catalase
H2O O2
Possible research questions, what are you going to investigate (independent variable)?
What is the effect of substrate concentration?
What is the effect of temperature?
What is the effect of pH?
Important things to consider:
How are you going to vary the mass/volume/concentration of your variable?
What units will you be measuring your variable in?
Have you chosen an effect range or values to answer your question?
Are the concentrations/chemicals you are using safe to handle?

56. How are you going to measure your results (dependent variable)?
Design of experiments to test the effect of temperature, pH and substrate concentration on the activity of enzymes. Experimental investigation of a factor affecting enzyme activity. (Practical 3)
How are you going to measure your results (dependent variable)?
What equipment will you be using to measure your results?
What are the units and uncertainty given both the equipment and how you choose to use it?
What time period do you need to run the experiment for? How fast is the enzyme action likely to be?
How many repeats will you need to make sure your results are reliable?

57. How are you going to make sure it is a fair test (control variables)?
Design of experiments to test the effect of temperature, pH and substrate concentration on the activity of enzymes. Experimental investigation of a factor affecting enzyme activity. (Practical 3)
How are you going to make sure it is a fair test (control variables)?
What variables other than your independent variable could affect the results?
Why would these variables affect the results?
How will you ensure each is kept constant and monitored?
What level should they be kept constant at? If a control variable is too far from it’s optimum then it could limit the enzyme action and no change would be seen in the results.
If a variable cannot be controlled it should still be discussed and considered as an uncontrolled variable.
Safety and ethics:
Are you using any equipment that may cause you or others harm? What steps have you taken to minimize this risk?

58. Calculating and plotting rates of reaction from raw experimental results.

63. Lab
Enzyme catalysis