1. Principles of Biochemistry
Horton • Moran • Scrimgeour • Perry • Rawn
Principles of Biochemistry
Fourth Edition
Chapter 6
Mechanisms of Enzymes
Prentice Hall c2002
Chapter 6
Copyright © 2006 Pearson Prentice Hall, Inc.

2. Chapter 6 Mechanisms of Enzymes
Mechanisms - the molecular details of catalyzed reactions
Enzyme mechanisms deduced from:
Kinetic experiments
Protein structural studies
Studies of nonenzymatic model systems
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3. 6.1 The Terminology of Mechanistic Chemistry
A reaction mechanism describes the details of a reaction
Reactants, products and intermediates are identified
Enzymatic mechanisms use the same symbolism used in organic chemistry
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4. Nucleophilic substitution reactions
Nucleophilic species are electron rich, and electrophilic species are electron poor
Types of nucleophilic substitution reactions include:
(1) Formation of a tetrahedral intermediate by nucleophilic substitution
(2) Direct displacement via a transition state
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5. Two types of nucleophilic substitution reactions
Formation of a tetrahedral intermediate
Direct displacement
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6. Cleavage reactions Carbanion formation Carbocation formation
Free radical formation
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7. Oxidation-reduction reactions
Electrons are transferred between two species
Oxidizing agent gains electrons (is reduced)
Reducing agent donates electrons (is oxidized)
Formaldehyde is oxidized by oxygen (below)
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8. 6.2 Catalysts Stabilize Transition States
Fig 6.1 Energy diagram for a single-step reaction
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9. Fig 6.2 Energy diagram for reaction with intermediate
Intermediate occurs in the trough between the two transition states
Rate determining step in the forward direction is formation of the first transition state
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10. Enzymes lower the activation energy of a reaction
(1) Substrate binding
Enzymes properly position substrates for reaction (makes the formation of the transition state more frequent and lowers the energy of activation)
(2) Transition state binding
Transition states are bound more tightly than substrates (this also lowers the activation energy)
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11. Fig 6.3 Enzymatic catalysis of the reaction A+B A-B
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12. 6.3 Chemical Modes of Enzymatic Catalysis
A. Polar Amino Acid Residues in Active Sites
Active-site cavity of an enzyme is lined with hydrophobic amino acids
Polar, ionizable residues at the active site participate in the mechanism
Anions and cations of certain amino acids are commonly involved in catalysis
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13. Table 6.1
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14. Table 6.2 pKa Values of amino acid ionizable groups in proteins
Group pKa
Terminal a-carboxyl 3-4
Side-chain carboxyl 4-5
Imidazole
Terminal a-amino
Thiol
Phenol
e-Amino ~10
Guanidine ~12
Hydroxymethyl ~16
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16. B. Acid-Base Catalysis
Reaction acceleration is achieved by catalytic transfer of a proton
A general base (B:) can act as a proton acceptor to remove protons from OH, NH, CH or other XH
This produces a stronger nucleophilic reactant (X:-)
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17. General base catalysis reactions (continued)
A general base (B:) can remove a proton from water and thereby generate the equivalent of OH- in neutral solution
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18. Proton donors can also catalyze reactions
A general acid (BH+) can donate protons
A covalent bond may break more easily if one of its atoms is protonated (below)
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19. C. Covalent Catalysis
All or part of a substrate is bound covalently to the enzyme to form a reactive intermediate
Group X can be transferred from A-X to B in two steps via the covalent ES complex X-E
A-X + E X-E + A
X-E + B B-X + E
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20. Sucrose phosphorylase exhibits covalent catalysis
Step one: a glucosyl residue is transferred to enzyme
*Sucrose + Enz Glucosyl-Enz + Fructose
Step two: Glucose is donated to phosphate
Glucosyl-Enz + Pi Glucose 1-phosphate + Enz
*(Sucrose is composed of a glucose and a fructose)
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21. D. pH Affects Enzymatic Rates
Catalytic mechanisms are dependent upon the state of ionization of catalytic groups
Plots of reaction velocity versus pH can indicate important ionizable catalytic groups
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22. Fig 6.4 pH-rate profile for papain
The two inflection points approximate the pKa values of the two ionizable residues
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23. Fig 6.5
Papain’s activity depends upon ionizable residues: His-159 and Cys-25
(a) Ribbon model (b) Active site residues (N blue, S yellow)
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24. Fig. 6.6
Three ionic forms of papain. Only the upper tautomer of the middle pair is active
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25. 6.4 Diffusion-Controlled Reactions
Enzyme rates can approach the physical limit of the rate of diffusion of two molecules in solution
Under physiological conditions the encounter frequency is about 108 to 109 M-1s-1
A few enzymes have rate-determining steps that are roughly as fast as the binding of substrates to the enzymes
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26. Table 6.4
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27. A. Triose Phosphate Isomerase (TPI)
TPI catalyzes a rapid aldehyde-ketone interconversion
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28. Role of active site residues in TPI catalysis
TPI has two ionizable active-site residues
Glu acts as a general acid-base catalyst
His shuttles protons between oxygens of an enzyme-bound intermediate
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29. Fig 6.7 Proposed mechanism for TPI
General acid-base catalysis mechanism (4 slides)
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30. Fig 6.7 TPI mechanism (continued)
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31. Fig 6.7 TPI mechanism (continued)
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32. Fig 6.7 TPI mechanism (continued)
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33. Figure 6.8
Structure of yeast (Saccharomyces cerevisiae) triose phosphate isomerase. The location of the substrate is indicated by the space-filling model of a transition state analogue. The side chains of the catalytic residues are represented by stick models: Glu-165 (red), His-95 (purple). [PDB 2YPI]
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34. Fig 6.9 Energy diagram for the TPI reaction
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35. B. Superoxide Dismutase
Superoxide dismutase is a very fast catalyst because: (1) The negatively charged substrate is attracted by a positively charged electric field near the active site (2) The reaction itself is very fast
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36. Two step reaction of superoxide dismutase
An atom of copper bound to the enzyme is reduced and then oxidized
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37. Figure 6.10
Surface charge on human superoxide dismutase. The structure of the enzyme is shown as a model that emphasizes the surface of the protein. Positively charged regions are colored blue and negatively charged regions are colored red. The copper atom at the active site is green. Note that the channel leading to the binding site is lined with hydrophilic residues. [PDB 1HL5]
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38. 6.5 Binding Modes of Enzymatic Catalysis
Proper binding of reactants in enzyme active sites provides substrate specificity and catalytic power
Two catalytic modes based on binding properties can each increase reaction rates over 10,000-fold :
(1) Proximity effect - collecting and positioning substrate molecules in the active site
(2) Transition-state (TS) stabilization - transition states bind more tightly than substrates
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39. Binding forces utilized for catalysis
1. Charge-charge interactions
2. Hydrogen bonds
3. Hydrophobic interactions
4. Van der Waals forces
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40. A. The Proximity Effect
Correct positioning of two reacting groups (in model reactions or at enzyme active sites):
(1) Reduces their degrees of freedom
(2) Results in a large loss of entropy
(3) The relative enhanced concentration of substrates (“effective molarity”) predicts the rate acceleration expected due to this effect
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41. Effective molarity k1(s-1) Effective molarity = k2(M-1s-1)
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42. Fig 6.11 Reactions of carboxylates with phenyl esters
Increased rates are seen when the reactants are held more rigidly in proximity (continued next slide)
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43. Fig (continued)
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44. B. Weak Binding of Substrates to Enzymes
Energy is required to reach the transition state from the ES complex
Excessive ES stabilization would create a “thermodynamic pit” and mean little or no catalysis
Most Km values (substrate dissociation constants) indicate weak binding to enzymes
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45. Fig 6.12 Energy of substrate binding
If an enzyme binds the substrate too tightly (dashed profile), the activation barrier (2) could be similar to that of the uncatalyzed reaction (1)
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46. C. Induced Fit
Induced fit activates an enzyme by substrate-initiated conformation effect
Induced fit is a substrate specificity effect, not a catalytic mode
Hexokinase mechanism requires sugar-induced closure of the active site
Glucose + ATP Glucose 6-phosphate + ADP
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47. Fig 6.13 Stereo views of yeast hexokinase
Yeast hexokinase contains 2 domains connected by a hinge region. Domains close on glucose binding.
(a) Open conformation
(b) Closed conformation
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48. D. Transition-State (TS) Stabilization
An increased interaction of the enzyme and substrate occurs in the transition-state (ES‡)
The enzyme distorts the substrate, forcing it toward the transition state
An enzyme must be complementary to the transition-state in shape and chemical character
Enzymes may bind their transition states 1010 to 1015 times more tightly than their substrates
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49. Transition-state (TS) analogs
Transition-state analogs are stable compounds whose structures resemble unstable transition states
Fig Phosphoglycolate, a TS analog for the enzyme triose phosphate isomerase
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50. Fig 6.15 Inhibition of adenosine deaminase by a TS analog
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51. Catalytic antibodies Fig 6.16 Antibody-catalyzed amide hydrolysis
(a) TS analog antigen
(b) Reaction catalyzed by the catalytic antibody
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52. 6.6 Lysozyme Binds an Ionic Intermediate Tightly
Lysozyme binds polysaccharide substrates (the sugar in subsite D of lysozyme is distorted into a half-chair conformation)
Binding energy from the sugars in the other subsites provides the energy necessary to distort sugar D
Lysozyme binds the distorted transition-state type structure strongly
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53. Fig 6.17 Bacterial cell-wall polysaccharide
Lysozyme cleaves bacterial cell wall polysaccharides (a four residue portion of a bacterial cell wall with lysozyme cleavage point is shown below)
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54. Fig 6.18 Lysozyme from chicken with a triaccharide molecule (pink)
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55. Fig 6.19 Conformations of N-acetylmuramic acid
(a) Chair conformation
(b) D-Site sugar residue is distorted into a higher energy half-chair conformation
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56. Lysozyme reaction mechanisms
1. Proximity effects
2. Acid-base catalysis
3. TS stabilization (or substrate distortion toward the transition state)
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57. Fig 6.20 Mechanism of lysozyme (2 slides)
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58. Prentice Hall c2002
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59. 6.7 Properties of Serine Proteases
A. Zymogens Are Inactive Enzyme Precursors
Digestive serine proteases including trypsin, chymotrypsin, and elastase are synthesized and stored in the pancreas as zymogens
Storage of hydrolytic enzymes as zymogens prevents damage to cell proteins
Zymogens are activated by selective proteolysis
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60. Fig 6.21 Activation of some pancreatic zymogens
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61. Fig 6.22 Stereo view of the backbones of chymotrypsinogen and a-chymotrypsin
Chymotrypsinogen (blue), a-chymotrypsin (green) Catalytic Asp, His, Ser (red), Ile-16, Asp-194 (yellow)
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62. B. Substrate Specificity of Serine Proteases
Many digestive proteases share similarities in 1o,2o and 3o structure
Chymotrypsin, trypsin and elastase have a similar backbone structure
Active site substrate specificities differ due to relatively small differences in specificity pockets
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63. Fig 6.23 Comparison of the backbones of (a) chymotrypsin (b) trypsin and (c) elastase
Backbone conformations and active-site residues (red) are similar in these three enzymes
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64. Fig 6.24 Binding sites of chymotrypsin, trypsin, and elastase
Substrate specificities are due to relatively small structural differences in active-site binding cavities
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65. C. Serine Proteases Use Both the Chemical and Binding Modes of Catalysis
a-Chymotrypsin active site groups include:
Ser identified by covalent DFP labeling
His-57 - identified by affinity labeling with TosPheCH2Cl
Asp identified by X-ray crystallography
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66. Fig 6.25 Stereo view of the catalytic site of chymotrypsin
Active-site Asp-102, His-57, Ser-195 are arrayed in a hydrogen-bonded network (O red, N blue)
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67. Fig 6.26 Catalytic triad of chymotrypsin
Imidazole ring (His-57) removes H from Ser-195 hydroxyl to make it a strong nucleophile (-CH2O-)
Buried carboxylate (Asp-102) stabilizes the positively-charged His-57 to facilitate serine ionization
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68. Fig 6.27 a-Chymotrypsin mechanism (8 slides)
Step (1): E + S
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69. Fig 6.27 (E-S)
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70. Fig 6.27 (E-TI1)
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71. Fig 6.27 (Acyl E + P1)
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72. Fig 6.27 (Acyl E + H2O)
(4)
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73. Fig 6.27 (E-TI2)
(5)
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74. Fig 6.27 (E-P2)
(6)
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75. Fig 6.27 (E + P2)
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