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

2. ENZYMES BASICS 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 globular shape A complex 3-D structure Human pancreatic 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. The Lock and Key Hypothesis Enzyme may be used again Enzyme-substrate complex E S P E E P Reaction coordinate

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

17. Factors affecting Enzymes substrate concentration pH temperature inhibitors

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

19. 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 V max will change too. Reaction velocity Substrate concentration V max

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

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. The effect of temperature Temperature / °C Enzyme activity 0 10 2030 4050 Q10 Denaturation

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. The effect of enzyme inhibition 1.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. Enzyme inhibitor complex Reversi ble reaction E + I EI

29. The effect of enzyme inhibition Succin ate Fumarate + 2H + + 2e - Succinate dehydrogenase CH 2 CO OH CHCO OH COO H CH 2 Malonate

30. The effect of enzyme inhibition 2.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 cytochrome oxidase. 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. ENZYMES KINETICS 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–Menten equation 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 E 0 : The parameters are k 2, the catalytic constant, and K m, the Michaelis constant.

35. Michaelis-Menten Kinetics

36. Enzyme Kinetics Enzymatic reaction E + S ESE + P k 1 k -1 k2k2 Rate expression for product formation v = dP/dt = k 2 (ES) d(ES)/dt = k 1 (E)(S)-k -1 (ES)-k 2 (ES) Conservation of enzyme (E) = (E 0 ) – (ES)

37. Two Methods to Proceed Rapid equilibrium assumption: define equilibrium coefficient K’ m = k -1 /k 1 = [E][S]/[ES] Quasi-steady state assumption [ES] = k 1 [E][S]/(k -1 +k 2 ) Both methods yield the same final equation

38. Michaelis- Menten Kinetics

39. When v= 1/2 V max, [S]= K m so Km is sometimes called the half-saturation constant and sometimes the Michaelis constant

40. Michaelis-Menten Kinetics units on k 2 are amount product per amount of enzyme per unit time (also called the “turnover number”). Units on E 0 are amount of enzyme (moles, grams, units, etc.) per unit volume K m has the same units as [S] (mole/liter, etc.)

41. Experimentally Determining Rate Parameters for Michaelis-Menten Kinetics Lineweaver-Burk Eadie-Hofstee Hanes- Woolf Batch 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. –Rewrite Michaelis-Menten rate expression –Plot 1/v versus 1/[S]. Slope is K m /V max, intercept is 1/V max Lineweaver-Burk (double reciprocal plot)

45. Graphical Solution 1/ V 1/ [S] 1/ V max -1/ K m Slope = K m / V max intercepts

46. Example: Lineweaver-Burk

47. Resulting Plot slope = K m / V max = 0.5686 y intercept = 1/ V max = 2.8687

48. Michaelis-Menten Kinetics

49. V max = 1/2.8687 x 10 -4 = 3.49 x 10 -5 M/min K m = 0.5686 x V m = 1.98 x 10 -5 M

50. Other Methods Eadie-Hofstee plot Hanes- Woolf

51. Comparison of Methods Lineweaver-Burk: supposedly gives good estimate for V max, error is not symmetric about data points, low [S] values get more weight Eadie-Hofstee: less bias at low [S] Hanes-Woolf: more accurate for V max. When trying to fit whole cell data – I don’t have much luck with any of them!

52. Batch Kinetics integrate rearrange

53. Inhibited Enzyme Kinetics Competitive Inhibition Noncompetitive Inhibition Uncompetitive Inhibition Substrate Inhibition

54. Effects of Temperature and pH

55. Experiments: Initial rate at different substrate concentrations ES 1 = 20ES 2 =10ES 4 =5ES 3 =6.7ES 5 =4 Measure S for a short time period. Calculate v from: v = [S(time 0) – S(time 1)]/delta time

56. Experiment Using S 1 Time (min) S (g/L) 0 20 0.519.43 v= (20-19.3)g/L]/0.5 min = 1.14 g/L/min Time (min) S (g/L) 0 10 0.5 9.565 v= (10-9.565)g/L]/0.5 min = 0.87 g/L/min Experiment Using S 2

57. Experimental Data S(mmol/L)v (mmol/L/min) 201.14 100.87 6.70.70 5.00.59 4.00.50 Problems with this method? Rate is not measured at a constant substrate concentration – substrate decreasing. Must have sensitive assay for substrate to measure initial rates.

59. Allosteric Enzyme Kinetics In an enzyme with more than one substrate binding site, binding of one substrate molecule affects the binding of another. n>1, cooperation; n<1, interference

60. Allosteric Enzymes Shape of rate curve is sigmoidal Michaelis-Menten Allosteric

61. Inhibition of Enzymes Can be irreversible (metals) or reversible (product, substrate, salt, etc.) 1.Competitive 2.Noncompetitive 3.Uncompetitive

62. Competitive Inhibition Inhibitor is an analog of the substrate, and binds to the active site of the enzyme. What assumption have we make in defining the parameters on the right?

63. Competitive Inhibition

64. Rate is given by: What is the magnitude of K m,app relative to K m and what will be the effect on v? How could you run a process to minimize the effects of this type of inhibition?

65. Competitive Inhibition 1/v V max is unchanged I > 0 I=0 1/V max 1/[S]-1/K m,app -1/K m

66. Practice deriving kinetic expressions Derive competitive inhibition equation (3.22 in your text)? Write down all assumptions.

67. Noncompetitive Inhibition Inhibitor binds to the enzyme, but not at the active site. However, the enzyme affinity for substrate is reduced.

68. Noncompetitive Inhibition

69. Cofactors and Coenzymes Holoenzymes- three parts Apoenzyme- Protein portion Cofactor- inorganic ion (ex: metal ions), improve the fit of enzyme with substrate Coenzyme- nonprotein organic molecule (ex: NAD- nicotinamide adenine dinucleotide), many synthesized from vitamins (why vitamins are essential)

70. Rate is given by: Noncompetitive Inhibition Question: What is the magnitude of V max,app relative to V max, and what will be the effect of v? How can you moderate the effects of this type of inhibition.

71. Noncompetitive Inhibition 1/v K m is unchanged I > 0 I=0 1/V max 1/[S] -1/K m 1/V max,app

72. Uncompetitive Inhibition Inhibitor binds only to ES complex, and not to E alone.

73. Uncompetitive Inhibition Rate is given by: What is the magnitude of V max,app relative to V max ? What is the magnitude of K m,app relative to K m ?

74. Uncompetitive Inhibition 1/v I > 0 I=0 1/V max 1/[S]-1/K m 1/V max,app -1/K m,app

75. Substrate Inhibition No substrate inhibition Substrate inhibition S v

76. Enzyme Deactivation Enzymes are denatured by –Temperature –pH –Radiation –Irreversible binding by inhibitors Temperature can both increase (thermal activation) and decrease (thermal denaturation) rate

77. Temperature effects At moderate temperatures, higher temperatures give higher rates. At higher temperatures, rate starts to decrease as enzyme denatures faster

78. Temperature Effects Effect on rate is a combination of the two effects Activation energy  10 kcal/mol Deactivation energy  100 kcal/mol