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Advanced Bioprocess Engineering Enzymes & Enzymes Kinetics PowerPoint Presentation
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Advanced Bioprocess Engineering Enzymes & Enzymes Kinetics

Advanced Bioprocess Engineering Enzymes & Enzymes Kinetics

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Advanced Bioprocess Engineering Enzymes & Enzymes Kinetics

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  1. Advanced Bioprocess EngineeringEnzymes &Enzymes Kinetics Lecturer Dr. Kamal E. M. Elkahlout Assistant Prof. of Biotechnology

  2. EnzymesBasics and Introduction

  3. ENZYMES A protein with catalytic properties due to its power of specific activation

  4. Chemical reactions • Chemical reactions need an initial input of energy = THE ACTIVATION ENERGY • During this part of the reaction the molecules are said to be in a transition state.

  5. Reaction pathway

  6. Making reactions go faster • Increasing the temperature make molecules move faster • Biological systems are very sensitive to temperature changes. • Enzymes can increase the rate of reactions without increasing the temperature. • They do this by lowering the activation energy. • They create a new reaction pathway “a short cut”

  7. An enzyme controlled pathway • Enzyme controlled reactions proceed 108 to 1011 times faster than corresponding non-enzymic reactions.

  8. Enzyme structure • Enzymes are proteins • They have a globularshape • A complex3-D structure Humanpancreatic amylase

  9. The active site • One part of an enzyme, the active site, is particularly important • The shape and the chemical environment inside the active site permits a chemical reaction to proceed more easily

  10. Cofactors • An additional non-protein molecule that is needed by some enzymes to help the reaction • Tightly bound cofactors are called prosthetic groups • Cofactors that are bound and released easily are called coenzymes • Many vitamins are coenzymes Nitrogenase enzyme with Fe, Mo and ADP cofactors )

  11. The substrate • The substrate of an enzyme are the reactants that are activated by the enzyme • Enzymes are specific to their substrates • The specificity is determined by the active site

  12. The Lock and Key Hypothesis • Fit between the substrate and the active site of the enzyme is exact • Like a key fits into a lock very precisely • The key is analogous to the enzyme and the substrate analogous to the lock. • Temporary structure called the enzyme-substrate complex formed • Products have a different shape from the substrate • Once formed, they are released from the active site • Leaving it free to become attached to another substrate

  13. S E E E Enzyme may be used again Enzyme-substratecomplex P P Reaction coordinate The Lock and Key Hypothesis

  14. The Lock and Key Hypothesis • This explains enzyme specificity • This explains the loss of activity when enzymes denature

  15. The Induced Fit Hypothesis • Some proteins can change their shape (conformation) • When a substrate combines with an enzyme, it induces a change in the enzyme’s conformation • The active site is then moulded into a precise conformation • Making the chemical environment suitable for the reaction • The bonds of the substrate are stretched to make the reaction easier (lowers activation energy)

  16. Hexokinase (a) without (b) with glucose substrate http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/ENZYMES/enzyme_mechanism.html The Induced Fit Hypothesis • This explains the enzymes that can react with a range of substrates of similar types

  17. Factors affecting Enzymes • substrate concentration • pH • temperature • inhibitors

  18. Reactionvelocity Substrate concentration Substrate concentration: Non-enzymic reactions • The increase in velocity is proportional to the substrate concentration

  19. Vmax Reactionvelocity Substrate concentration Substrate concentration: Enzymic reactions • Faster reaction but it reaches a saturation point when all the enzyme molecules are occupied. • If you alter the concentration of the enzyme then Vmax will change too.

  20. Enzyme activity Trypsin Pepsin 5 11 9 3 1 7 pH The effect of pH Optimum pH values

  21. The effect of pH • Extreme pH levels will produce denaturation • The structure of the enzyme is changed • The active site is distorted and the substrate molecules will no longer fit in it • At pH values slightly different from the enzyme’s optimum value, small changes in the charges of the enzyme and it’s substrate molecules will occur • This change in ionisation will affect the binding of the substrate with the active site.

  22. The effect of temperature • Q10 (the temperature coefficient) = the increase in reaction rate with a 10°C rise in temperature. • For chemical reactions the Q10 = 2 to 3(the rate of the reaction doubles or triples with every 10°C rise in temperature) • Enzyme-controlled reactions follow this rule as they are chemical reactions • BUT at high temperatures proteins denature • The optimum temperature for an enzyme controlled reaction will be a balance between the Q10 and denaturation.

  23. Denaturation Q10 Enzyme activity 10 0 20 30 40 50 Temperature / °C The effect of temperature

  24. The effect of temperature • For most enzymes the optimum temperature is about 30°C • Many are a lot lower, cold water fish will die at 30°C because their enzymes denature • A few bacteria have enzymes that can withstand very high temperatures up to 100°C • Most enzymes however are fully denatured at 70°C

  25. Inhibitors • Inhibitors are chemicals that reduce the rate of enzymic reactions. • The are usually specific and they work at low concentrations. • They block the enzyme but they do not usually destroy it. • Many drugs and poisons are inhibitors of enzymes in the nervous system.

  26. The effect of enzyme inhibition • Irreversible inhibitors: Combine with the functional groups of the amino acids in the active site, irreversibly. Examples: nerve gases and pesticides, containing organophosphorus, combine with serine residues in the enzyme acetylcholine esterase.

  27. The effect of enzyme inhibition • Reversible inhibitors: These can be washed out of the solution of enzyme by dialysis. There are two categories.

  28. E + I EI Enzyme inhibitor complex Reversible reaction The effect of enzyme inhibition • Competitive: These compete with the substrate molecules for the active site. The inhibitor’s action is proportional to its concentration. Resembles the substrate’s structure closely.

  29. Fumarate + 2H++ 2e- Succinate Succinate dehydrogenase CH2COOH CHCOOH COOH CH2 CH2COOH CHCOOH COOH Malonate The effect of enzyme inhibition

  30. The effect of enzyme inhibition • Non-competitive: These are not influenced by the concentration of the substrate. It inhibits by binding irreversibly to the enzyme but not at the active site. Examples • Cyanide combines with the Iron in the enzymes cytochromeoxidase. • Heavy metals, Ag or Hg, combine with –SH groups. These can be removed by using a chelating agent such as EDTA.

  31. Applications of inhibitors • Negative feedback: end point or end product inhibition • Poisons snake bite, plant alkaloids and nerve gases. • Medicine antibiotics, sulphonamides, sedatives and stimulants

  32. EnzymesKinetics of Enzyme Reactions

  33. INTRODUCTION The objectives of studying kinetics: 1) Gain an understanding of the mechanisms of enzyme action; 2) Illuminate the physiological roles of enzyme-catalyzed reactions 3) Manipulate enzyme properties for biotechnological ends.

  34. MICHAELIS–MENTEN KINETICS Michaelis–Mentenequation expresses the initial rate (v) of a reaction at a concentration (S) of the substrate transformed in a reaction catalyzed by an enzyme at total concentration E0: The parameters are k2, the catalytic constant, and Km, the Michaelisconstant.

  35. Michaelis-Menten Kinetics

  36. k1 k-1 k2 Enzyme Kinetics Enzymatic reaction E + S ES E + P Rate expression for product formation v = dP/dt = k2(ES) d(ES)/dt = k1(E)(S)-k-1(ES)-k2(ES) Conservation of enzyme (E) = (E0) – (ES)

  37. Two Methods to Proceed • Rapid equilibrium assumption: define equilibrium coefficient K’m = k-1/k1 = [E][S]/[ES] • Quasi-steady state assumption [ES] = k1[E][S]/(k-1+k2) • Both methods yield the same final equation

  38. Michaelis- Menten Kinetics

  39. Michaelis-Menten Kinetics • When v= 1/2 Vmax, [S]= Km so Km is sometimes called the half-saturation constant and sometimes the Michaelis constant

  40. Michaelis-Menten Kinetics • units on k2 are amount product per amount of enzyme per unit time (also called the “turnover number”). Units on E0 are amount of enzyme (moles, grams, units, etc.) per unit volume • Km has the same units as [S] (mole/liter, etc.)

  41. Experimentally Determining Rate Parameters for Michaelis-Menten KineticsLineweaver-Burk Eadie-Hofstee Hanes- WoolfBatch Kinetics

  42. Determining Parameters • Rearrange the equation into a linear form. • Plot the data. • What kind of data would we have for an experiment examining enzyme kinetics? • Describe an experiment. • The intercept and slope are related to the parameter values.

  43. Enzyme Kinetics Experiment Place enzyme and substrate (reactants) in a constant temperature, well stirred vessel. Measure disappearance of reactant or formation of product with time. Why constant temperature? Why well stirred? What about the medium? Buffer?

  44. Lineweaver-Burk (double reciprocal plot) • Rewrite Michaelis-Menten rate expression • Plot 1/v versus 1/[S]. Slope is Km/Vmax, intercept is 1/Vmax

  45. intercepts Graphical Solution 1/ V Slope = Km/ Vmax 1/ Vmax 1/ [S] -1/ Km

  46. Example: Lineweaver-Burk

  47. Resulting Plot slope = Km/ Vmax= 0.5686 y intercept = 1/ Vmax= 2.8687

  48. Michaelis-Menten Kinetics

  49. Vmax = 1/2.8687 x 10-4 = 3.49 x 10-5 M/min Km= 0.5686 x Vm = 1.98 x 10-5 M

  50. Other Methods • Eadie-Hofstee plot • Hanes- Woolf