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CU 0304 Enzymes

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  1. 1. ENZYMES
  2. 2. Define the following terms:1. Anabolic reactions: Reactions that build up molecules2. Catabolic reactions: Reactions that break down molecules Combination of anabolic and catabolic3. Metabolism: reactions4. Catalyst: A substance that speeds up reactions without changing the produced substances5. Metabolic pathway: Sequence of enzyme controlled reactions6. Specificity: Only able to catalyse specific reactions7. Substrate: The molecule(s) the enzyme works on8. Product: Molecule(s) produced by enzymes
  3. 3. Naming enzymes:• Intracellular enzymes Work inside cells eg.DNA polymerase• Extracellular enzymes Secreted by cells and work outside cells eg. pepsin, amylase• Recommended names Short name, often ending in ‘ase’ eg. creatine kinase• Systematic name Describes the type of reaction being catalysed eg. ATP:creatine phosphotransferase• Classification number Eg.
  4. 4. Timeline of enzyme discovery 1835: Breakdown of starch to sugar by malt 1877: Name enzyme coined to describe chemicals in yeast that ferment sugars 1897:Eduard Buchner extracted enzyme from yeast and showed it could work outside cells 1905: Otto Rohm exyracted pancreatic proteases to supply enzymes for tanning 1926: James B Sumner produced first pure crystalline enzyme (urease) and showed enzymes were proteins 1930-1936: Protein nature of enzymes finally established when digestive enzymes crystallised by John H Northrop 1946: Sumner finally awarded Nobel prize
  5. 5. What Are Enzymes?• Most enzymes are Proteins (tertiary and quaternary structures)• Act as Catalyst to accelerates a reaction• Not permanently changed in the process 5
  6. 6. Enzymes are globular proteins• Active site has a specific shape due to tertiary structure of protein.• A change in shape of the protein affects shape of active site and the function of the enzyme.
  7. 7. How do enzymes Work?Enzymes work by weakening bonds which lowers activation energy 7
  8. 8. Energy levels of molecules Enzymes lower the activation energy of a reaction Activation energy of uncatalysed Initial energy state Activation energy reactions of substrates of enzyme catalysed reaction Final energy state of products Progress of reaction (time)
  9. 9. Enzymes lower activation energy by forming an enzyme/substrate complex Substrate + Enzyme Enzyme/substrate complex Enzyme/product complex Product + Enzyme
  10. 10. Lock and Key
  11. 11. Lock-and-key hypothesis assumes the active site of an enzyme is rigid in its shapeHow ever crystallographic studies indicate proteins are flexible.
  12. 12. Enzyme-Substrate ComplexThe substance (reactant) an enzyme acts on is the substrate Joins Substrate Enzyme 12
  13. 13. In anabolic reactionsenzymes bring the substratemolecules together.In catabolic reactions theenzyme active site affectsthe bonds in substrates sothey are easier to break
  14. 14. Active Site• A restricted region of an enzyme molecule which binds to the substrate. substrate Active Site Substrate Enzyme 14
  15. 15. Induced Fit• A change in the shape of an enzyme’s active site• Induced by the substrate 15
  16. 16. Induced Fit• A change in the configuration of an enzyme’s active site (H+ and ionic bonds are involved).• Induced by the substrate. Active Site substrate Enzyme induced fit 16
  17. 17. Induced Fit
  18. 18. The Induced-fit hypothesis suggests the active site isflexible and only assumes its catalytic conformation afterthe substrate molecules bind to the site. When the product leaves the enzyme the active site reverts to its inactive state.
  19. 19. Enzyme reactionsenzyme + substrate enzyme-substrate complex E +S ES
  20. 20. Enzyme reactions enzyme + substrate enzyme-substrate complex E +S ESenzyme-substrate complex enzyme + product ES E +P
  21. 21. Characteristics of enzymes• Only change the rate of reaction. They do not change the equilibrium or end products.• Specific to one particular reaction• Present in very small amounts due to high molecular activity: Turnover number = number of substrate molecules transformed per minute by one enzyme molecule Catalase turnover number = 6 x106/min
  22. 22. Enzymes in medicineGlucose oxidase + peroxidase + blue dye on dipsticks todetect glucose in urine: Glucose oxidase Glucose Hydrogen peroxide peroxidase Dye: Blue---Green---Brown Dye changes according to amount of glucose Enzyme-linked immunosorbent assays (ELISAs) detect antibodies to infections.
  23. 23. What Affects Enzyme Activity?• Three factors: 1. Environmental Conditions 2. Cofactors and Coenzymes 3. Enzyme Inhibitors 23
  24. 24. 1. Environmental Conditions1. Extreme Temperature are the mostdangerous- high temps may denature (unfold) theenzyme.2. pH (most like 6 - 8 pH near neutral)3. Ionic concentration (salt ions) 24
  25. 25. 2. Cofactors and Coenzymes• Inorganic substances (zinc, iron) and vitamins (respectively) are sometimes need for proper enzymatic activity. activity• Example: Iron must be present in the quaternary structure - hemoglobin in order for it to pick up oxygen. 25
  26. 26. Two examples of Enzyme Inhibitorsa. Competitive inhibitors: are chemicals that resemble an enzyme’s normal substrate and compete with it for the active site. site Substrate Enzyme Competitive inhibitor 26
  27. 27. Inhibitorsb. Noncompetitive inhibitors: Inhibitors that do not enter the active site, but site bind to another part of the enzyme causing the enzyme to change its shape, which in turn shape alters the active site. siteSubstrate Noncompetitive Enzyme Inhibitor active site altered 27
  28. 28. Enzyme activityHow fast an enzyme is working Rate of Reaction
  29. 29. Enzyme activity How fast an enzyme is working Rate of ReactionRate of Reaction = Amount of substrate changed (or amount product formed) in a given period of time.
  30. 30. Enzyme activityRate of Reaction Variable you are looking at
  31. 31. Enzyme activityFour Variables
  32. 32. Enzyme activity Temperature pHFour Variables Enzyme Concentration Substrate Concentration
  33. 33. Rate of Reaction Temperature
  34. 34. TemperatureRate of Reaction 0 10 20 30 40 50 60
  35. 35. 5- 40oC Temperature Increase in Activity 40oC - denatures Rate of Reaction 0 10 20 30 40 50 60<5oC - inactive
  36. 36. Effect of heat on enzyme activty If you heat the protein above its optimal temperature bonds breakmeaning the protein loses it secondary and tertiary structure
  37. 37. Effect of heat on enzyme activty Denaturing the protein
  38. 38. Effect of heat on enzyme activty Denaturing the protein ACTIVE SITE CHANGES SHAPE SO SUBSTRATE NO LONGER FITSEven if temperature lowered – enzyme can’t regain its correct shape
  39. 39. Rate of Reaction pH
  40. 40. Rate of Reaction12345 pH6789
  41. 41. pH Narrow pH optimaRate of Reaction 1 2 3 4 5 6 7 8 9
  42. 42. pH Narrow pH optimaRate of Reaction WHY? 1 2 3 4 5 6 7 8 9
  43. 43. pH Narrow pH optimaRate of Reaction Disrupt Ionic bonds - Structure Effect charged residues at active site 1 2 3 4 5 6 7 8 9
  44. 44. Enzyme ConcentrationRate of Reaction
  45. 45. Enzyme ConcentrationRate of Reaction Enzyme Concentration
  46. 46. Substrate Concentration Rate of Reaction
  47. 47. Substrate Concentration Rate of Reaction Substrate Concentration
  48. 48. Substrate Concentration Active sites full- maximum turnover Rate of Reaction Substrate Concentration
  49. 49. How would you measure the effect of an enzyme?• Compare uncatalysed rate with catalysed.• Enzymes can increase rate by a factor of between 108 to 1026
  50. 50. Characteristics of enzymes• Rate of enzyme action is dependent on number of substrate molecules present Vmax = maximum rate of reaction Rate of Reaction (M) Vmax approached as all active sites become filled Some active sites free at lower substrate concentrations Substrate concentration
  51. 51. Why do scientists measure the initial rate of reaction of enzyme-catalysed reactions? Initial rate of reaction Rate of Reaction (M) They measure rate at start of reaction before any factors, eg. substrate concentration, have had time to change. Independent variable
  52. 52. Rate of enzyme –catalysed reactions are affectedby temperature.Temperature coefficient Q10: rate of reaction at (x + 10) oC Q10 = ----------------------------------------- rate of reaction at x oC Q10 for between 0 - 40 oC is 2
  53. 53. Enzymes denature at 60oC Rate of reaction Optimum temperature Rate doubles Enzyme denaturing and every 10oC losing catalytic abilities TemperatureSome thermophilic bacteria have enzymes with optimumtemperatures of 85oC
  54. 54. pH affects the formation of hydrogen bonds and sulphur bridges in proteins and so affects shape. trypsin cholinesterase pepsinRate of Reaction (M) 2 4 6 8 10 pH