1. Enzymes: increase the rates of reactions are highly specific for their preferred substrate Can be regulated can be localized in certain organelles Can be organized into pathways.

2. Rate Enhancement Enzyme Non-enzymic t 1/2 (yr) Rate Enhancement OMP decarboxylase 78 000 0001.4 X 10 17 Adenosine deaminase 1202.1 X 10 12 Cytidine deaminase 691.2 X 10 12 Carbonic anhydrase 5 sec7.7 X 10 6

3. The Thermodynamics of a Reaction Free Energy (G) Progress of the reaction Substrate (1 M) Reaction performed Under standard conditions: 25 o C and 1 atmosphere air pressure

4. The Thermodynamics of a Reaction Free Energy (G) Progress of the reaction Substrate (1 M) Product (1 M) Reaction performed Under standard conditions: 25 o C and 1 atmosphere air pressure G0G0

5. The Thermodynamics of a Reaction Free Energy (G) Progress of the reaction Substrate (1 M) Product (1 M) Reaction performed Under standard conditions: 25 o C and 1 atmosphere air pressure G0G0 A negative  G 0 Makes the reaction Thermodynamically favourable

6. The Thermodynamics of a Reaction Free Energy (G) Progress of the reaction Substrate (1 M) Product (1 M) Reaction performed Under standard conditions: 25 o C and 1 atmosphere air pressure G0G0 The  G o is related to the K eq. The exact relationship is as follows:  G o = -RTlnK eq

7. The Kinetics of a Reaction Free Energy (G) Progress of the reaction Substrate (1 M) Product (1 M) G0G0

8. The Kinetics of a Reaction Free Energy (G) Progress of the reaction Substrate (1 M) Product (1 M) G0G0 Activation Energy  G #

9. The Kinetics of a Reaction Free Energy (G) Progress of the reaction Substrate (1 M) Product (1 M) G0G0 The enzyme lowers the activation energy  G #

10. The Kinetics of a Reaction Free Energy (G) Progress of the reaction Substrate (1 M) Product (1 M) G0G0 The enzyme lowers the activation energy  G #

11. Recapping… Enzymes CANNOT change the thermodynamics of a reaction:  G o or K eq They CANNOT change the direction of a reaction or the position of the equilibrium. They DO increase the rate of the reaction by lowering the activation energy.

12. Reaction Rate Measurements The rate of a reaction is measured as the #moles of product produced per unit time. The most user friendly units are  mol/min. The term ASSAY is used in Biochemistry to describe a reaction that measures something; enzyme activity or the concentration of a metabolite.

13. Measuring the rate of a reaction Time (min) [product]

14. Measuring the rate of a reaction Time (min) [product] dP/dt The initial linear rate Is used for all enzyme kinetics measurements

15. Calculating Velocity Using the Alkaline Phosphatase Experiment, suppose you had a change in Absorbance per min (  A/min) of 0.3….. –Step 1 Convert to  concentration/min by dividing the  A/min by the extinction coefficient (  ), say 15 mM -1 cm -1 giving  conc/min = 0.02 mM/min –Step 2 Convert to #nmoles/min in the assay. The assay is 1 mL so 0.02 mM/min = 0.02  mol/mL/min = 20 nmol/min/assay.

16. The Effect of [substrate] Reaction rate [substrate] on a simple first order reaction without an enzyme Slope = k, the rate constant First order means the reaction rate is dependent on the concentration of only one reactant.

17. The Effect of [substrate] Reaction rate [substrate] on a simple first order reaction with an enzyme All the available enzyme is saturated with substrate

18. The Effect of [substrate] Reaction rate [substrate] on a simple first order reaction with an enzyme Vmax KMKM The K M is the [S] at ½V max

19. The Effect of [substrate] Reaction rate [substrate] on a simple first order reaction with an enzyme Vmax KMKM The K M is the [S] at ½V max The K M describes the shape of the curve

20. Vmax: What is the maximum speed the car can go at?

21. K M : how much petrol do you need to travel at 60 kph? Maybe the little car is more efficient?

22. The Lineweaver-Burk Plot: A double reciprocal plot used to find Vmax and K M 1/Vmax -1/K M 1/v 1/[S]

23. The K M Reaction rate [substrate] Vmax KMKM KMKM Two different isoenzymes with different K M s for the same substrate. Which has the higher affinity for the substrate?

24. The K M Reaction rate [substrate] Vmax KMKM KMKM Higher affinity because it takes less substrate to attain Vmax.

25. The K M Reaction rate [substrate] Vmax KMKM KMKM Higher affinity means a lower K M

26. The Progress of the Reaction in more detail. E + SESEX # E + P Free Energy (G) Progress of the reaction S P + E G0G0 EX # ES G#G#

27. The K M and the Vmax E + SESE + P KMKM Kcat = Vmax/[E] Measures the affinity of the enzyme and substrate Measures how fast the reaction can go

28. The K M Rate: ES E + S Rate: E + S ES K M = OR Rate of dissociation Rate of association K M = In most cases:

29. The Significance of K M  The [S] which gives ½ V max  A measure of the affinity the enzyme has for the substrate  A low K M means high affinity and vice versa a high K M means low affinity  The K M is independent of the [E]

30. What is Vmax? Vmax is measured in Units (U). 1 Unit (U) is the amount of enzyme required to release 1  mole of product (P) in 1 minute under Vmax conditions. You measure the rate of the reaction over a short time (min).

31. Vmax and [E] Vmax [enzyme] The Vmax can be used practically to measure the amount of active enzyme in a sample e.g. serum. You will use this in the gene expression prac.

32. k cat The number of molecules of substrate converted to product by 1 molecule of enzyme in 1 second. This is equivalent to the #  moles of product produced per sec per  mole of enzyme OR the #nmoles P/sec/nmol E Units are seconds -1

33. Calculating k cat Begin with the Vmax In the Alkaline Phosphatase experiment the Vmax is usually around 30 nmol/min. This works out to 0.5 nmoles P/sec (/60) Now all we need to know is how many nmoles of Enzyme produced this rate. To calculate this we need to know how much enzyme we added to the assay and the molecular weight of the enzyme.

34. Calculating k cat In the Alkaline Phosphatase experiment you added 20  L of enzyme solution containing 50  g/mL enzyme. This means we have 20*50 ng = 1  g. So now our Vmax rate is: 0.5 nmoles P/sec/1 000 ng E

35. Calculating k cat The Vmax rate is: 0.5 nmoles P/sec/1 000 ng E If the molecular weight of Alkaline Phosphatase is 100,000 then there is 1/100 (1 000/100 000) of a nmole of Enzyme in the assay.

36. Calculating k cat If 1/100 th of a nmole of Enzyme catalyses the formation of 0.5 nmoles of product in 1 second then…. 1 nmole of enzyme will catalyse 0.5*100 = 50 nmoles of product in 1 second The K cat = 50 nmol sec -1 nmol -1 The K cat = 50 sec -1

37. The Steady State Assumption Used to explain the shape of the hyperbola A level of ES, [ES], is established very early in the reaction This [ES] level is dependent on the [S] This [ES] remains constant throughout the reaction.

38. The Steady State Assumption Time (min) [conc] Substrate Product

39. The Steady State Assumption Total Enzyme added = [ES] + [Efree] [ES] Time (min) [conc] [Enzyme free ]

40. The Michaelis Menten Plot Reaction rate [substrate] Vmax KMKM The K M is the [S] at ½V max

41. Prac Results: Alkaline Phosphatase

44. The Michaelis Menten Relationship The equation below describes the hyperbola. Velocity = Vmax * [S] ([S] + Km)

45. Deriving the Michaelis Menten Relationship The steady state assumption means that the rate of formation of ES = the rate of breakdown of ES E + S ESE + P k1k1 k2k2 K- 1

46. Deriving the Michaelis Menten Relationship Mathematically the steady state assumption means that the rate of formation of ES = the rate of breakdown of ES…. Rate of formation = k 1 *[E]*[S] Rate of breakdown = k -1 *[ES] + k 2 *[ES] Therefore: k 1 *[E]*[S] = k -1 *[ES] + k 2 *[ES] E + S ESE + P k1k1 k2k2 K- 1

47. Deriving the Michaelis Menten Relationship k 1 *[E]*[S] = k -1 *[ES] + k 2 *[ES] Simplifying k 1 *[E]*[S] = [ES] (k -1 + k 2 ) Now K M = (k -1 + k 2 )/ k 1 So [E]*[S] = [ES]* K M E + S ESE + P k1k1 k2k2 K- 1

48. Deriving the Michaelis Menten Relationship [E]*[S] = [ES]* K M Now the Total amount of Enzyme E T = E free + ES  E = E free = E T – ES  [E T ] – [ES] = [ES]* K M /[S]  [E T ] = [ES]* K M /[S] + [ES] E + S ESE + P k1k1 k2k2 K- 1

49. Deriving the Michaelis Menten Relationship  [E T ] = [ES]* K M /[S] + [ES] Now Vmax = [E T ]*k 2 and velocity (v) = [ES]*k 2 So multiplying both sides by k 2 [E T ]* k 2 = [ES]* k 2 (K M /[S] + 1) Vmax = v *(K M /[S] + 1) E + S ESE + P k1k1 k2k2 K- 1

50. Deriving the Michaelis Menten Relationship Vmax = v *(K M +[S])/[S] ) Cross multiplying 1/v = (K M + [S]) Inverting V = Vmax * [S] (K M +[S]) E + S ESE + P k1k1 k2k2 K- 1 Vmax*[S]

51. k cat /K M This measurement reflects the efficiency of the enzyme. What we really want to know is how often ES goes to E + P. An efficient enzyme will send all the ES to E + P. An inefficient enzyme will send some back to E + S

52. k cat /K M K cat is k 2 and K M = (k -1 + k 2 )/k 1 So k cat /K M = k 2 * k 1 / (k -1 + k 2 ) Now if the enzyme is really efficient k -1 will be really small (very little ES going back to E + S) k cat /K M approaches k 1. This is limited by the rate of diffusion. E + S ESE + P k1k1 k2k2 K- 1

53. Inhibitors Inhibitors can be irreversible or reversible. Irreversible inhibitors usually covalently bind to the enzyme; they are often slower to act (time dependent inhibition) and present as non-competitive inhibition. They cannot be dialysed out or diluted out. Reversible inhibitors; competitive, non- competitive, mixed, uncompetitive

54. The Competitive Inhibitor The inhibitor binds to the same site on the enzyme as the substrate Thus it competes with the substrate It eventually reaches Vmax It needs more substrate to do it  Km increases

56. The Effect of a Competitive Inhibitor Reaction rate [substrate] Vmax KMKM The competitive Inhibitor Is in yellow

57. The Competitive Inhibitor 1/Vmax -1/K M 1/v 1/[S] No Inhibitor A Competitive Inhibitor

58. The Competitive Inhibitor 1/Vmax -1/K M 1/v 1/[S] No Inhibitor A Competitive Inhibitor Vmax unchanged Km increases -1/Km increases

59. The Non-Competitive Inhibitor The inhibitor binds to a site other than the substrate binding site Both I and S can bind to the enzyme simultaneously It never reaches Vmax The Km does not change It is like adding less enzyme to an assay

61. The Effect of a Non-competitive Inhibitor Reaction rate [substrate] Vmax KMKM The non-competitive Inhibitor Is in yellow

62. The Non-competitive Inhibitor: 1/Vmax -1/K M 1/v 1/[S] No Inhibitor A Non-competitive Inhibitor

63. The Non-competitive Inhibitor: 1/Vmax -1/K M 1/v 1/[S] No Inhibitor A Non-competitive Inhibitor Vmax decreases 1/Vmax increases Km unchanged

64. The mixed Non-Competitive Inhibitor The inhibitor binds to a site other than the substrate binding site But the binding of the substrate to the enzyme alters the affinity of the inhibitor for the enzyme Both Km and Vmax change The most common type of inhibition

65. K i ≠ K’ i

66. The mixed non-competitive Inhibitor: 1/Vmax -1/K M 1/v 1/[S] No Inhibitor A mixed non-competitive Inhibitor

67. The Uncompetitive Inhibitor The inhibitor binds to the ES complex only Both Km and Vmax decrease Vmax/Km unchanged

69. The Uncompetitive Inhibitor: 1/Vmax -1/K M 1/v 1/[S] No Inhibitor An uncompetitive Inhibitor Vmax decreases Km decreases Vmax/Km unchanged

70. Regulatory Enzymes Allosteric regulation: the binding of a small molecule (ligand) distant from the active site, Covalent modification, often phosphorylation of serine, threonine or tyrosine residues,

71. Regulatory Enzymes Changes in the amount of enzyme either by changes in gene expression, (often at the level of transcription) or protein turnover, Substrate availability, Inhibition (competitive etc), Activation of zymogens or pro- enzymes.

72. Allosteric Regulation

73. Allosteric Enzymes Substrate (blue triangle) binds to the first subunit. This makes it easier for the next substrate molecule to bind Ultimately the whole enzyme is occupied by substrate.

74. Allosteric Regulation [S] Velocity a b c

75. Covalent Modification Usually phosphorylation Residues: serine, threonine or tyrosine The example of glycogen synthase Often used in cell signalling Insulin signalling

76. Zymogen Activation Often used with proteases To prevent random destruction of the cell Produced as an inactive form A small portion cleaved off to activate Chymotrypsinogen  chymotrypsin Trypsinogen  trypsin