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Enzyme kinetics and associated reactor design: Determination of the kinetic parameters of

CP504 – ppt_Set 03. Enzyme kinetics and associated reactor design: Determination of the kinetic parameters of enzyme-induced reactions. learn about the meaning of kinetic parameters learn to determine the kinetic parameters

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Enzyme kinetics and associated reactor design: Determination of the kinetic parameters of

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  1. CP504 – ppt_Set 03 Enzyme kinetics and associated reactor design: Determination of the kinetic parameters of enzyme-induced reactions • learn about the meaning of kinetic parameters • learn to determine the kinetic parameters • learn the effects of pH, temperature and substrate concentration on enzyme activity (or reaction rates) • learn about inhibited enzyme kinetics • learn about allosteric enzymes and their kinetics

  2. Simple Enzyme Kinetics (in summary) k1 k3 E + S ES E + P k2 which is equivalent to [E] P S S for substrate (reactant) E for enzyme ES for enzyme-substrate complex P for product

  3. Simple Enzyme Kinetics (in summary) [E] P S rmaxCS = - rS rP = KM + CS wherermax = k3CE0= kcatCE0 and KM = f(rate constants) rmax is proportional to the initial concentration of the enzyme KM is a constant

  4. rmaxCS = - rS KM + CS Simple Enzyme Kinetics (in summary) -rs Catalyzed reaction Catalyzed reaction rmax rmax uncatalyzed reaction 2 KM Cs

  5. An exercise Consider an industrially important enzyme, which catalyzes the conversion of a protein substrate to form a much more valuable product.  The enzyme follows the Briggs-Haldane mechanism: An initial rate analysis for the reaction in solution, with E0 = 0.10 μM and various substrate concentrations S0, yields the following Michaelis-Menten parameters: Vmax = 0.60 μM/s; KM = 80 μM. A different type of experiment indicates that the association rate constant, k1, is k1 = 2.0 x 106 M-1s-1 (2.0 μM-1s-1). a. Estimate the values of k2 and k-1. b. On average, what fraction of enzyme-substrate binding events result in product formation? Source: Jason Haugh, Department of Chemical & Biomolecular Engineering, North Carolina State University

  6. Simple Enzyme Kinetics (in summary) Catalytic step k1 k3 E + S ES E + P k2 Substrate binding step k3 = kcat

  7. learn about the meaning of kinetic parameters • learn to determine the kinetic parameters • learn the effects of pH, temperature and substrate concentration on enzyme activity (or reaction rates) • learn about inhibited enzyme kinetics • learn about allosteric enzymes and their kinetics

  8. How to determine the kinetic parameters rmax and KM ? Carry out an enzyme catalysed experiment, and measure the substrate concentration (CS) with time. rmaxCS - rS = KM + CS

  9. How to determine the M-M kinetics rmax and KM ? Carry out an enzyme catalysed experiment, and measure the substrate concentration (CS) with time. rmaxCS - rS = KM + CS

  10. CS KM 1 CS + = - rS rmax rmax 1 KM 1 1 + = - rS rmax rmax CS - rS rmax - - rS KM = CS We could rearrange rmaxCS - rS = KM + CS to get the following 3 linear forms: (14) (15) (16)

  11. CS - rS 1 rmax The Langmuir Plot CS KM 1 CS (14) + = - rS rmax rmax - KM CS

  12. CS - rS 1 rmax The Langmuir Plot CS KM 1 CS (14) + = - rS rmax rmax • Determine rmax more accurately than the other plots. - KM CS

  13. 1 - rS KM rmax 1 CS The Lineweaver-Burk Plot 1 KM 1 1 (15) + = - rS rmax rmax CS 1 - KM

  14. 1 - rS KM rmax 1 CS The Lineweaver-Burk Plot 1 KM 1 1 (15) + = - rS rmax rmax CS • Gives good estimates of rmax, but not necessarily KM • - Data points at low substrate concentrations influence the slope and intercept more than data points at high Cs 1 - KM

  15. KM -rS CS The Eadie-Hofstee Plot - rS rmax - - rS KM = (16) CS - rS rmax KM

  16. KM -rS CS The Eadie-Hofstee Plot - rS rmax - - rS KM = (16) CS - rS • Can be subjected to large errors since both coordinates contain (-rS) • - Less bias on point at low Cs than with Lineweaver-Burk plot rmax KM

  17. Data: Determine the M-M kinetic parameters for all the three methods discussed in the previous slides.

  18. rmax = 1 / slope = 1 / 1.5866 = 0.63 mmol/l.min KM = rmax x intercept = 0.63 x 4.6417 = 2.93 mmol/l

  19. rmax = 1 / intercept = 1 / 1.945 = 0.51 mmol/l.min KM = rmax x slope = 0.51 x 3.4575 = 1.78 mmol/l

  20. rmax = intercept = 0.54 mmol/l.min KM = - slope = 1.89 mmol/l

  21. Comparison of the results

  22. Comparison of the results

  23. Comparison of the results

  24. learn about the meaning of kinetic parameters • learn to determine the kinetic parameters • learn the effects of pH, temperature and substrate concentration on enzyme activity (or reaction rates) • learn about inhibited enzyme kinetics • learn about allosteric enzymes and their kinetics http://www.youtube.com/watch?v=D2j2KGwJXJc

  25. Effects of temperature on enzyme activity: • Increases in the temperature of a system results from increases in the kinetic energy of the system. • Kinetic energy increase has the following effects on the rates of reactions: • More energetic collisions • Increase in the number of collisions per unit time • Denaturation of the enzyme or substrate http://academic.brooklyn.cuny.edu/biology/bio4fv/page/enz_act.htm

  26. Effects of temperature on enzyme activity: More energetic collisions: When molecules collide, the kinetic energy of the molecules can be converted into chemical potential energy of the molecules. If the chemical potential energy of the molecules become great enough, the activation energy of a exergonic reaction can be achieved and a change in chemical state will result. Thus the greater the kinetic energy of the molecules in a system, the greater is the resulting chemical potential energy when two molecules collide. As the temperature of a system is increased it is possible that more molecules per unit time will reach the activation energy. Thus the rate of the reaction may increase. http://academic.brooklyn.cuny.edu/biology/bio4fv/page/enz_act.htm

  27. Effects of temperature on enzyme activity: Increase in the number of collisions per unit time: In order to convert substrate into product, enzymes must collide with and bind to the substrate at the active site. Increasing the temperature of a system will increase the number of collisions of enzyme and substrate per unit time. Thus, within limits, the rate of the reaction will increase. http://academic.brooklyn.cuny.edu/biology/bio4fv/page/enz_act.htm

  28. Effects of temperature on enzyme activity: Denaturation of the enzyme: Enzymes are very large proteins whose three dimensional shape is vital for their activity. When proteins are heated up too much they vibrate. If the heat gets too intense then the enzymes literally shake themselves out of shape, and the structure breaks down. The enzyme is said to be denatured. A denatured enzyme does not have the correct 'lock' structure. Therefore it cannot function efficiently by accepting the 'key' substrate molecule. http://www.woisd.net/moodle/mod/resource/view.php?id=44

  29. Effects of temperature on enzyme activity: Denaturation of the enzyme:

  30. Effects of temperature on enzyme activity: Denaturation of the enzyme: As temperature increases, enzyme activity increases until its optimum temperature is reached. At higher temperatures, the enzyme activity rapidly falls to zero.

  31. Effects of temperature on enzyme activity: Denaturation for most human enzymes: The optimum temperature for most human enzymes to work at is around 37ºC which is why this temperature is body temperature. Enzymes start to denature at about 45°C. Optimal for most human enzymes http://www.woisd.net/moodle/mod/resource/view.php?id=44

  32. Effects of temperature on enzyme activity: Optimal for most human enzymes Optimal for some thermophillic bacterial enzymes Reaction rate Temperature (deg C) https://wikispaces.psu.edu/display/230/Enzyme+Kinetics+and+Catalysis

  33. Effects of pH on enzyme activity: The structure of the protein enzyme can depends on how acid or alkaline the reaction medium is, that is, it is pH dependent. If it is too acid or too alkaline, the structure of the protein is changed and it is 'denatured' and becomes less effective. If the enzyme does not have the correct 'lock' structure, it cannot function efficiently by accepting the 'key' substrate molecule. In the optimum pH range, the enzyme catalysis is at its most efficient.

  34. Effects of pH on enzyme activity: Optimal for trypsin (an intestinal enzyme) Optimal for pepsin (a stomach enzyme) Reaction rate pH https://wikispaces.psu.edu/display/230/Enzyme+Kinetics+and+Catalysis

  35. Effects of pH on enzyme activity: Amylase (pancreas) enzyme Optimum pH: 6.7 - 7.0 Function: A pancreatic enzyme that catalyzes the breakdown/hydrolysis of starch into soluble sugars that can readily be digested and metabolised for energy generation. Amylase (malt) enzyme Optimum pH: 4.6 - 5.2 Function: Catalyzes the breakdown/hydrolysis of starch into soluble sugars in malt carbohydrate extracts. www.docbrown.info/page01/ExIndChem/ExIndChema.htm

  36. Effects of pH on enzyme activity: Catalase enzyme Optimum pH: ~7.0 Function: Catalyses the breakdown of potentially harmful hydrogen peroxide to water and oxygen. Important in respiration/metabolism chemistry. 2H2O2(aq)==> 2H2O(l)+ O2(g) www.docbrown.info/page01/ExIndChem/ExIndChema.htm

  37. Effects of pH on enzyme activity: Invertase enzyme Optimum pH: 4.5 Function: Catalyses the breakdown/hydrolysis of sucrose into fructose + glucose, the resulting mixture is 'inverted sugar syrup'. C12H22O11 + H2O ==> C6H12O6 + C6H12O6 www.docbrown.info/page01/ExIndChem/ExIndChema.htm

  38. Effects of pH on enzyme activity: Lipase (pancreas) enzyme Optimum pH: ~8.0 Function: Lipases catalyse the breakdown dietary fats, oils, triglycerides etc. into digestible molecules in the human digestion system. Lipase (stomach) enzyme Optimum pH: 4.0 - 5.0 Function: As above, but note the significantly different optimum pH in the acid stomach juices, to optimum pH in the alkaline fluids of the pancreas. www.docbrown.info/page01/ExIndChem/ExIndChema.htm

  39. Effects of pH on enzyme activity: Maltase enzyme Optimum pH: 6.1 - 6.8 Function: Breaks down malt sugars. www.docbrown.info/page01/ExIndChem/ExIndChema.htm

  40. Effects of pH on enzyme activity: Pepsin enzyme Optimum pH: 1.5 - 2.0 Function: Catalyses the breakdown/hydrolysis of proteins into smaller peptide fragments. www.docbrown.info/page01/ExIndChem/ExIndChema.htm

  41. Effects of pH on enzyme activity: Trypsin enzyme Optimum pH: 7.8 - 8.7 Function: Catalyses the breakdown/hydrolysis of proteins into amino acids. Note again, the significantly different optimum pH to similarly functioning pepsin. www.docbrown.info/page01/ExIndChem/ExIndChema.htm

  42. Effects of pH on enzyme activity: Urease enzyme Optimum pH: ~7.0 Function: Catalyzes the breakdown of urea into ammonia and carbon dioxide. (NH2)2(aq)+ H2O(l) ==> 2NH3(aq)+ CO2(aq) www.docbrown.info/page01/ExIndChem/ExIndChema.htm

  43. Effects of substrate concentration on enzyme activity: www.docbrown.info/page01/ExIndChem/ExIndChema.htm

  44. Effect of shear

  45. Complex enzyme kinetics • learn about the meaning of kinetic parameters • learn to determine the kinetic parameters • learn the effects of pH, temperature and substrate concentration on enzyme activity (or reaction rates) • learn about inhibited enzyme kinetics • learn about allosteric enzymes and their kinetics

  46. Inhibited enzyme reactions Inhibitors are substances that slow down the rate of enzyme catalyzed reactions. There are two distinct types of inhibitors: - Irreversible inhibitors form a stable complex with enzymes and reduce enzyme activity (e.g. lead, cadmium, organophosphorous pesticide) - Reversible inhibitors interact more loosely with enzymes and can be displaced.

  47. Inhibited enzyme reactions - applications Many drugs and poisons are inhibitors of enzymes in the nervous system. Poisons: snake bite, plant alkaloids and nerve gases Medicines: antibiotics, sulphonamides, sedatives and stimulants

  48. Primary constituents of Snake Venom Enzymes - Spur physiologically disruptive or destructive processes. Proteolysins - Dissolve cells and tissue at the bite site, causing local pain and swelling. Cardiotoxins - Variable effects, some depolarise cardiac muscles and alter heart contraction, causing heart failure. Harmorrhagins - Destroy capillary walls, causing haemorrhages near and distant from the bite. Coagulation - Retarding compounds prevent blood clotting. Thromboses - Coagulate blood and foster clot formation throughout the circulatory system. Haemolysis - Destroy red blood cells. Cytolysins - Destroy white blood cells. Neurotoxins - Block the transmission of nerve impulses to muscles, especially those associated with the diaphragm and breathing. http://www.writework.com/essay/biochemistry-snake-venom

  49. Inhibited enzyme reactions Inhibitors are also classified as competitive and non-competitive inhibitors.

  50. Competitive inhibition • - The structure of inhibitor molecule closely resembles the chemical structure and molecular geometry of the substrate. • - The inhibitor competes for the same active site as the substrate molecule. • It does not alter the structure of the enzyme. • The inhibitor may interact with the enzyme at the active site, but no reaction takes place. http://www.elmhurst.edu/~chm/vchembook/573inhibit.html

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