1. Unit F212: Molecules, biodiversity, food & health 2.1.3 Enzymes By Mr. Wilson

2. Enzymes All GLOBULAR proteins of tertiary structure, usually with HYDROPHOBIC amino acid R-groups in centre of the molecule and HYDROPHILIC ones around the outside. Tertiary structure (shape) gives an enzyme its properties by forming a receptor site (ACTIVE SITE ), which is usually an area of fewer than 10 amino acids and composed of their R-groups.

3. Enzymes Hydrogen bonds, ionic bonds and di- sulphide bonds hold the tertiary structure shape together. Act on SUBSTRATES to make PRODUCTS. Are biological catalysts & have many industrial uses as they are fast and don’t produce any unwanted by- products.

4. Properties of enzymes They are specific. One enzyme will only catalyse one type of reaction. Not used up in reactions. Only small amounts are needed to catalyse a lot of substrate. They have a high turnover number. Affected by pH and temperature changes.

5. Question: ?Why do temperature changes affect enzyme action?

6. Naming enzymes Enzymes are usually named after the substrate they act upon: Amylase acts on amylose (starch). Lactase acts on lactose. Protease acts on proteins. ATPase acts on ATP. Cellulase acts on cellulose. READ PAGES 122 to 123.

7. Where enzymes work best Essential to metabolic reactions in ALL types of organism. Endotherms regulate temperature & so their enzymes can function at optimum levels in a variety of environments. Can be EXTRACELLULAR (eg. digestion) or INTRACELLULAR (eg. catalysing reactions in the cytoplasm of cells).

8. Where enzymes work best

9. Phagocyte white blood cells use enzymes to digest microbes. READ PAGES 124 to 125.

10. Enzyme action: Lock & key theory

11. The small area of the enzyme that comes into contact with the substrate is called the active site. While substrate is attached to enzyme in the active site an enzyme/substrate complex is formed. Because products have a different shape they are released as they no longer fit.

12. Enzyme action: Induced-fit theory

13. Suggests the active site may not exactly fit the substrate, but is slightly flexible and can mould around the substrate with charges on active site amino acids also being important. The reaction only proceeds when tight bonding is achieved.

14. Enzyme action: Activation energy To break & reassemble important biological molecules (such as maltose + water > glucose + glucose) takes an additional input of energy. This is called the ACTIVATION ENERGY. Enzymes are able to lower activation energy & thus enzyme controlled reactions can proceed at much lower temperatures.

15. Enzyme action: Activation energy

16. Why can we not just increase the temperature in cells to speed up these reactions? Enzymes are able to carry out this function due to the specificity of the active site for its substrate molecule. READ PAGES 126 to 127.

17. Enzymes & temperature Enzymes & substrates collide & react. The number of collisions, and their force, is increased with an increase in temperature (kinetics). Heating enzyme controlled reactions therefore increases the rate of reaction, but only to the optimum temperature. For most enzymes this is around 40ºC, but it depends on an organism’s internal and external environment.

18. Enzymes & temperature Graphs show the rate of enzyme catalyzed reactions steadily increasing until the optimum temperature is reached. After this point the reaction slows considerably and at high temperatures the reaction stops altogether. At this point the enzyme’s tertiary structure has collapsed. We call this DENATURATION of the enzyme.

19. Enzymes & temperature

20. DENATURATION occurs because the increased heat gives increased kinetic energy to the atoms in the enzyme molecules. As they vibrate more & more vigorously weaker bonds such as H-bonds and Ionic bonds break, the tertiary structure is compromised and the active site is no longer the shape it should be. READ PAGES 128 to 129.

21. Enzymes & pH pH 1 to 6 is acidic and indicates the presence of positively charged Hydrogen ions (H + ). H + can interfere with the H-bonds and Ionic bonds that stabilise an enzyme’s tertiary structure and thus active site. This can, for example, ‘replace’ essential H-bonds. Increasing acidity will therefore alter the rate of the enzyme-controlled reaction.

22. Enzymes & pH Furthermore, the H + ions can alter the essential & specific charges on amino acid R-groups that make up the active site. This will reduce bonding and slow down the enzyme-controlled reaction. All enzymes have an optimum pH; the concentration of H + ions in the solution that give the enzyme it’s best shape, and thus active site shape, to carry out its function.

23. Enzymes & pH

24. The optimum pH gives the maximum rate of reaction. Most enzymes have a narrow pH range for the reasons outlined previously. Extremes of pH can DENATURE enzymes, but small changes can be tolerated (when pH returns to optimum, bonds can reform). READ PAGES 130 to 131.

25. Effect of substrate concentration With a fixed amount of enzyme, increases in substrate concentration will increase the rate of an enzyme catalysed reaction up to a point. When enzyme active sites are all fully occupied at all times and working at capacity the rate of reaction will level off and the reaction rate reaches a maximum value.

26. Effect of substrate concentration

27. Effect of enzyme concentration The number of substrate molecules an enzyme can turn into products in one minute is called the turnover number. Chymotrypsin – 6000 Catalase – 5600000 Carbonic anhydrase – 36000000 With unlimited substrate, optimum pH and optimum temperature, rate of reaction will be directly proportional to enzyme concentration.

28. Effect of enzyme concentration

29. If the concentration of substrate is fixed however, increasing the enzyme concentration will increase the rate of reaction to a certain point before reaching a maximum. In this situation active sites will begin to remain empty and thus the reaction rate cannot continue to increase. READ PAGES 132 to 133.

30. Focus on: Investigating enzymes It is very common to investigate the action of Catalase (usually sourced from liver or potato) on hydrogen peroxide. This is because oxygen gas is a product; its volume can be measured. It’s also common to investigate the effect that changing the enzyme or substrate concentration has on the rate of this reaction.

31. Focus on: Investigating enzymes You may remember doing this by some method at GCSE. There are other ways to investigate the rate of reaction using this enzyme & substrate too. Watch the demonstrations. The measurements for concentration of H 2 O 2 are complicated so often arbitrary (made up, but in scale) units are used.

32. Enzymes: review

33. Enzymes at work: Inhibitors Can reduce the rate of enzyme-controlled reactions. Combine with enzymes and in some way stop them attaching substrate. Can be specific or affect a range of enzymes. Can be reversible and non-reversible (permanent). When a reversible inhibitor is removed from an enzyme it will work effectively again. Reversible inhibitors can be competitive and non-competitive.

34. Enzymes at work: Inhibitors Competitive reversible inhibitors have a substrate- similar structure and so fill active sites stopping substrate from attaching. ROR slows until inhibitor is removed. Increasing the substrate concentration will increase the rate of reaction. Explain why. Example; Malonate is a competitive reversible inhibitor of succinate in the active site of succinate dehydrogenase, an enzyme in respiration.

35. Enzymes at work: Inhibitors Non-competitive reversible inhibitors attach to a site on the enzyme other than the active site and change the tertiary structure (shape) of the enzyme so that its active site changes shape and substrate can no longer bind. Concentration of substrate has no effect on the rate of reaction in this case. Explain why.

36. Enzymes at work: Inhibitors Most competitive inhibitors are reversible (they are temporarily in the active site). Many non-competitive inhibitors are permanent (irreversible/non- reversible) inhibitors that alter enzyme shape permanently so that they will no longer work at all. You could say the enzyme is effectively DENATURED by them. Examples: Heavy metal ions such as Silver & Mercury are non-competitive, non-reversible inhibitors, breaking disulphide bridges. Potassium cyanide is a non-competitive, non-reversible inhibitor of cytochrome oxidase in the essential metabolic process; respiration, and as such can be a deadly poison. READ PAGES 134 to 135.

37. Focus on: Investigating enzymes Copper sulphate is commonly used to investigate the effect of inhibitors on enzyme activity. This is because it yields copper ions which act as non-competitive, non-reversible inhibitors to a variety of enzymes. Increasing concentrations of copper sulphate are often used to demonstrate increasing levels of inhibition (slowed rate of enzyme- controlled reaction).

38. Enzymes at work: Cofactors Some enzymes require these additional substances to work effectively. They are all non-protein molecules. They act to modify the chemical structure of enzymes in a way that allows them to function at an appropriate rate. 3 types – prosthetic groups, coenzymes & inorganic ion cofactors (activators).

39. Enzymes at work: Cofactors; coenzymes Coenzymes – small organic molecules that attach temporarily to the active site of an enzyme (just before or with the substrate). They are changed in the reaction, but are recycled to be used again. The enzyme can only work effectively when it is attached. Many are derived from vitamins. NAD is an example. It comes from nicotinic acid and is important for many dehydrogenase enzymes.

40. Enzymes at work: Cofactors; prosthetic groups Prosthetic groups are coenzymes that are permanently attached to an enzyme. They contribute to the shape, charge & properties of the enzyme. Other proteins have these too. The prosthetic haem group in haemoglobin allows it to carry oxygen. Hb could do this without it.

41. Enzymes at work: Cofactors; inorganic ion cofactors These may bind to substrate or enzyme and allow more effective binding to form the enzyme- substrate complex. They do this by changing the overall charge (they are ions) and sometimes the overall shape of the ES complex. Obtained from food/minerals/supplements. Examples: Mg 2+ is needed for the enzymes of protein synthesis to work. Amylase requires Cl - ions to work.

42. Homework. READ PAGES 136 to 137. Complete a short essay to explain the action of some poisons and drugs on enzyme activity. Start by addressing the info on pages 138 to 139. READ PAGES 144 to 145. Try to answer the Qs, in your head only, after reading.

43. Focus on: Investigating enzymes You will be asked to bring this presentation back in for a review lesson in the week beginning 16 th March ‘09. In preparation for the practical skills assessment: Read through pages 140 to 143. Answer Qs 1, 2 & 3 on page 141 and Qs 1 & 2 on page 143.