1. 1 Enzymes I Reactions, Kinetics, Inhibition, Applications Enzymes as Biological Catalysts The Kinetic Properties of Enzymes Substrate Binding and Enzyme Action Enzyme Inhibition Applications of Enzyme Action Enzymes as Biological Catalysts The Kinetic Properties of Enzymes Substrate Binding and Enzyme Action Enzyme Inhibition Applications of Enzyme Action

2. 2 Enzymes as biological catalysts Biological catalysts Typically are very large proteins. Permit reactions to ‘go’ at conditions that the body can tolerate. Can process millions of molecules every second. substrates Are very specific - react with one or only a few types of molecules (substrates).

3. 3 Introduction to enzymes Consider the following reaction: 2 H 2 O 2 2 H 2 O + O 2 The reaction is thermodynamically favored but occurs very slowly. Slow reaction rate is due to the high activation energy for the reaction. Only a small portion of the molecules have sufficient energy to overcome this energy. We could increase the energy of the system but this is not an option for biological systems.

4. 4 Energy diagram 2 H 2 O 2 2 H 2 O + O 2 H O HO HO HO Energy HH activation energy transition state reactants products

5. 5 Enzymatic reactions Enzymes act by providing an alternate, easier pathway for a reaction. Same reactants, products and equilibrium. Increase reaction rates by having a lower activation energy barrier. cofactor Some enzymes require an additional component to function properly - cofactor. This can be an organic or organometallic molecule or metal ion like Cu 2+, Zn 2+ or Mg 2+

6. 6 Enzymatic reactions Energy 2 H 2 O 2 HH 2 H 2 O + O 2 enzymatic activation energy

7. 7 Naming of enzymes Name is based on:- what it reacts with - how it reacts -ase - add -ase endingExamples lactase lactase - enzyme that reacts with lactose. pyruvate decarboxylase pyruvate decarboxylase - removes carboxyl from pyruvate. ase Each enzyme has an official name ending in ase and a four digit classification number

8. 8 Classification of enzymes Based on type of reaction Oxidoreductase Oxidoreductase catalyze a redox reaction Transferase Transferase transfer a functional group Hydrolase Hydrolase cause hydrolysis reactions Lyase Lyase break C-O, C-C or C-N bonds Isomerases Isomerases rearrange functional groups Ligase Ligase join two molecules

9. 9 Enzyme classes Absolutely specific Only reacts with a single substrate. Group specific Works with similar molecules with the same functional group. Linkage specific Catalyzes a specific combination of bonds. Stereochemically specific D-L- Only will work with the proper D- or L- form.

10. 10 The kinetic properties of enzymes For non-catalyzed reactions Reaction rate increase with concentration. Enzyme catalyzed reactions Also increase but only to a certain point. V max V max maximum velocity This catalytic behavior is observed for most enzymes.

11. 11 Effect of substrate concentration Rate of reaction (velocity) Substrate concentration A plot of initial reaction rates at various concentrations shows that a maximum reaction rate is observed if all other conditions are held constant. A plot of initial reaction rates at various concentrations shows that a maximum reaction rate is observed if all other conditions are held constant.

12. 12 Michaelis-Menten equation The mechanism for this type of reaction was originally formulated by Michaelis and Menten. In the simplest case, it involves the reaction of a substrate (S) with an enzyme (E) to initially form an activated complex (ES). The complex can then decompose to a product (P) and the enzyme or back to the substrate.

13. 13 Michaelis-Menten equation E + S ES P + E E + S E + P ES energy Although the presence of an enzyme will reduce the activation energy for a reaction, it does not eliminate it. As a result, product tends to accumulate.

14. 14 Michaelis-Menten equation E + S ES P + E Three rate expressions are used to describe the enzymatic reaction: rate f = k 1 [E o -ES][S]formation of ES rate d = k 2 [ES]decomposition of ES rate p = k 3 [ES]formation of product E o = initial enzyme concentration k 4 is neglected because its effect is very small during the initial stages of the reaction. k3k3 k2k2 k1k1 k4k4

15. 15 Michaelis-Menten equation Typically, as this type of reaction proceeds, it reaches an equilibrium like condition where [ES] remains constant. rate f = rate d + rate p If we substitute in our rate expressions and rearrange, we end up with: [ES] = k 1 [E o ] [S] (k 2 + k 3 )/k 1 + k1[S]

16. 16 Michaelis-Menten equation Michaelis constant - K m We can simplify our equation by including all of the rate constants in a single term. The rate of product formation is then: This is the step of greatest analytical interest. K m = k 2 + k 3 k 1 rate p = k 3 [E o ] [S] K M + [S]

17. 17 Michaelis-Menten equation Typically, it is the substrate that is to be measured so k 3 [E o ] will control the rate. Maximum velocity Maximum velocity - V max = k 3 [E o ] This represents the maximum attainable reaction rate based on the initial enzyme concentration. Our rate of product formation is then: rate p = V max [S] K M

18. 18 Michaelis-Menten equation Rate of reaction (velocity) Substrate concentration V max 1/2 V max KMKM K M = [S] where v = 1/2 V max

19. 19 Michaelis-Menten equation If k 2 is much greater than k 3 then: k 2 k 1 K M = In this form, K M is the dissociation constant for the ES complex. A large K M indicates that ES complex is held together rather weakly A small K M indicates that the forces holding the ES complex together are strong.

20. 20 Turnover number This is a measure of how rapidly an enzyme can process a substrate. turnover number = k 3 = V max [E T ] Example. Example. A 10 -9 M solution of catalase causes the breakdown of 0.4 M H 2 O 2 per second. k 3 = 0.4 moles/liter H 2 O 2 per second 10 -9 moles/liter catalase k 3 = 40,000,000 H 2 O 2 per mole of catalase per second

21. 21 Lineweaver-Burk equation Using the Michaelis-Menten equation can be difficult to determine V max from experimental data. An alternate approach was proposed by Lineweaver and Burk that results in a linear plot of data. =. + 1vo1vo K M V max 1 [S] 1 V max

22. 22 Lineweaver-Burk equation 1/v o 1 / [S]-1 / K M slope of line K M / V max y intercept 1 / V max

23. 23 Factors that influence enzyme activity Other conditions and species can alter the performance of an enzyme. Environmental factors Temperature, pHCofactors Metal ions, organic and organometallic speciesEffectors Species that alter enzyme activity

24. 24 Factors that influence enzyme activity Effect of pH on enzyme activity vovo 2 4 6 8 10 pH trypsin pepsin

25. 25 Examples of optimum pH Optimum EnzymeSourcepH pepsingastric mucosa 1.5 sucraseintestine 6.2 catalaseliver 7.3 arginasebeef liver9.0 alkalinebone9.5 phosphatase

26. 26 Effect of temperature on enzymatic reactions Exceeding normal temperature ranges always reduces enzyme reaction rates. temperature Optimum temperature is usually 25 - 40 o C but not always.

27. 27 Substrate binding and enzyme action

28. 28 Steps in an enzymatic reaction 1.Enzyme and substrate combine to form a complex. 2. Complex goes through a transition state - not quite substrate or product 3. A complex of the enzyme and the product is produced 4. Finally the enzyme and product separate All of these steps are equilibria. Lets review each step

29. 29 The players Catalytic site Binding site Substrate Enzyme

30. 30 Formation of the enzyme-substrate complex First step in an enzyme catalyzed reaction E + S ES Enzyme Substrate Complex

31. 31 Formation of the transition state An intermediate species is then formed. ES ES * scissile bond scissile bond

32. 32 Formation of the enzyme-product complex The enzyme-product complex is then formed. ES * EP

33. 33 Formation of the product The product is finally made and the enzyme is ready for another substrate. EP E + P

34. 34 The active site Enzymes are typically HUGE proteins, yet only a small part is actually involved in reaction. The active site has two basic components. catalytic site binding site Model of triose-p-isomerase Model of triose-p-isomerase

35. 35 Characteristics of enzyme active sites Catalytic site Where the reaction actually occurs. Binding site Area that holds substrate in proper place. Enzymes use weak, non-covalent interactions to hold the substrate in place based on R groups of amino acids. Shape is complementary to the substrate and determines the specificity of the enzyme. Sites are pockets or clefts on the enzyme surface.

36. 36 Characteristics of enzyme active sites Lock and key model 1890 picture by Emil Fisher. This model assumed that only a substrate of the proper shape could fit with the enzyme. Induced-fit model Proposed by Daniel Koshland in 1958. This model assumes continuous changes in active site structure as a substrate binds.

37. 37 Lock and key model This model assumes that an enzyme active site will only accept a specific substrate.

38. 38 Induced fit model This new model recognizes that there is much flexibility in an enzyme’s structure. According to the model, an enzyme is able to conform to a substrate.

39. 39 General acid-base catalysis Enzyme functional groups in the active site region can serve as acids (-COOH) or bases (-COO -, -NH 2 ).

40. 40 Metal-ion catalysts Metal ions associated with an enzyme or substrate often participate in catalysis. Common metal ions Common metal ions: Na +, K +, Mg 2+, Mn 2+, Cu 2+, Zn 2+, Fe 2+, Fe 3+, Ni 2+ They assist by one of the following actions. Properly holding substrate in place using coordinate covalent bonds Enhance a reaction by polarizing the scissile bond or stabilizing a negatively charged intermediate. Participate in an oxidation-reduction reaction.

41. 41 Covalent catalysis This occurs when a nucleophilic functional group on an enzyme reacts to form a covalent bond with the substrate. This leads to an intermediate form that is highly reactive. Serine proteases are a group of enzymes that rely on this approach.

42. 42 Covalent catalysis Step 1 Step 2 covalent intermediate

43. 43 Enzyme inhibition Many substances can inhibit enzyme activity. substrate analogs, toxins, drugs, metal complexes Inhibition studies can provide: Information on metabolic pathways. Insight on how drugs and toxins exert their effects. Better understanding of enzyme reaction mechanisms.

44. 44 Reversible and irreversible inhibitors Two broad classes of inhibitors have been identified based on the extent of interaction.Irreversible Forms covalent or very strong noncovalent bonds. The site of attack is an amino acid group that participates in the normal enzymatic reaction.Reversible Forms weak, noncovalent bonds that readily dissociate from an enzyme. The enzyme is only inactive when the inhibitor is present.

45. 45 Inhibitors Competitive inhibitor. Resembles the normal substrate and competes with it for the same site. normal substrate competitive inhibitor

46. 46 Inhibitors Noncompetitive inhibitors. Materials that bind at a location other than the normal site. This results in a change in how the enzyme performs. inhibitor noncompetitive site

47. 47 Inhibitors Uncompetitive inhibitor. Similar to a noncompetitive inhibitor but only binds to the ES complex.

48. 48 Acetylcholinesterase and nerve transmission This enzyme is needed to transmit a nerve signal at a neuromuscular junction. Arrival of a nerve signal causes Ca 2+ levels to increase. This causes acetylcholine containing vesicles to move to end of the nerve cell and is released. Acetylcholine then diffuses across synapse to pass the signal to the muscle. Acetylcholinesterase then destroys the acetylcholine to stop the signal.

49. 49 Acetylcholinesterase and nerve transmission acetylcholine receptor protein acetylcholine receptor protein Presence of acetylcholine at receptor causes a flow of sodium and potassium ions. This causes a muscle contraction. synaptic cleft synaptic cleft acetylcholinesterase - destroys excess acetylcholine acetylcholinesterase - destroys excess acetylcholine

50. 50 Acetylcholinesterase Stick model of acetylcholinesterase.

51. 51 Acetylcholinesterase and nerve transmission Without the enzyme, muscles would continue to contract causing spasms. Acetylcholinesterase inhibitors are used as drugs and poisons. Organo fluorophosphates Bind to the enzyme. Death can occur.Succinylcholine Acts like acetylcholine and binds to sites on the muscle. Used as a muscle relaxant.

52. 52 Analytical methods for determination of substrates Two approaches can be used. Add a large amount of enzyme and measure the product after complete reaction of the substrate. Not a good choice because enzymes are relatively expensive. Add a small amount of enzyme and determine the initial rate of reaction.

53. 53 Analytical methods for determination of substrates Other factors to consider. Temperature, pH and other conditions must be held constant. Other materials may compete for either your enzyme or substrate. These should be masked, removed or at least held constant. Any loss of products or interactions of the products with other materials must be addressed.

54. 54 Examples Determination of urea Based on the catalyzed hydrolysis of urea by urease. NH 2 CONH 2 + 2H 2 O + H + 2NH 4 + + HCO 3 - Potential species to measure H + NH 4 + HCO 3 - urease All are pH dependent

55. 55 Examples Determination of urea An easy approach would appear to be to measure the pH - using a pH electrode. Unfortunately, as H + is consumed, the reaction rate changes. pH stat pH stat - a device that monitors a solution and adds acid or base to keep pH constant. A plot of acid added at a fixed time verses [S] produces a linear relationship.

56. 56 Examples Determination of glucose. This is a common material to assay for in clinical laboratories. Enzymatic reaction used: glucose + H 2 O + O 2 gluconic acid + H 2 O 2 Two approaches are used for measure the rate based on measurement of O 2 or H 2 O 2. peroxidase

57. 57 Examples Determination of glucose - H 2 O 2 Peroxide can’t be measured directly by any rapid, convenient method. coupled reaction A coupled reaction is used to produce a detectable species. H 2 O 2 + reduced dye H 2 O + oxidized dye The increase in absorbance can be measured which is proportional to the concentration of glucose. peroxidase colorlesscolored

58. 58 Examples Determination of glucose - O 2 Polarographic method With this approach, [O 2 ] is directly measured at an electrode using the following reaction: O 2 + 4H + + 4e - = H 2 O A commercial glucose analyzer has been developed using this approach.

59. 59 Examples Determination of glucose - O 2 Polarographic method Only a VERY small amount of O 2 is actually used and it is VERY rapid (~10sec) so conditions don’t change very much. The sample must NOT be in rapid equilibrium with the atmosphere in order to get reliable results.