1. The Organic Chemistry of Enzyme-Catalyzed Reactions Revised Edition
Professor Richard B. Silverman
Department of Chemistry
Department of Biochemistry, Molecular Biology, and Cell Biology
Northwestern University

2. The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 1 Enzymes as Catalysts

3. For published data regarding any enzyme see:

4. What are enzymes, and how do they work?
First “isolation” of an enzyme in 1833
Ethanol added to aqueous extract of malt
Yielded heat-labile precipitate that was utilized to hydrolyze starch to soluble sugar; precipitate now known as amylase
Kühne coined term enzyme - means “in yeast”
Duclaux proposed all enzymes should have suffix “ase”

5. Enzymes - natural proteins that catalyze chemical reactions
First enzyme recognized as protein was jack bean urease
Crystallized in 1926
Took 70 more years (1995), though, to obtain its crystal structure

6. Enzymes have molecular weights of several thousand to several million, yet catalyze transformations on molecules as small as carbon dioxide and nitrogen
Function by lowering transition-state energies and energetic intermediates and by raising the ground-state energy
Many different hypotheses proposed for how enzymes catalyze reactions
Common link of hypotheses: enzyme-catalyzed reaction always initiated by the formation of an enzyme-substrate (or ES) complex in a small cavity called the active site

7. Lock-and-key hypothesis - Fischer proposed enzyme is the lock into which the substrate (the key) fits
Does not rationalize certain observed phenomena:
Compounds having less bulky substituents often fail to be substrates
Some compounds with more bulky substituents bind more tightly
Some enzymes that catalyze reactions between two substrates do not bind one substrate until the other one is bound

8. 1958 - Induced-fit hypothesis proposed by Koshland:
When a substrate begins to bind to an enzyme, interactions induce a conformational change in the enzyme
Results in a change of the enzyme from a low catalytic form to a high catalytic form
Induced-fit hypothesis requires a flexible active site

9. Concept of flexible active site stated earlier by Pauling (1946):
Hypothesized that an enzyme is a flexible template that is most complementary to substrates at the transition state rather than at the ground state
Therefore, the substrate does not bind most effectively in the ES complex
As reaction proceeds, enzyme conforms better to the transition-state structure
Transition-state stabilization results in rate enhancement

10. Only a dozen or so amino acid residues may make up the active site
Only two or three may be involved directly in substrate binding and/or catalysis

11. Why is it necessary for enzymes to be so large?
Most effective binding of substrate results from close packing of atoms within protein
Remainder of enzyme outside active site is required to maintain integrity of the active site
May serve to channel the substrate into the active site
Active site aligns the orbitals of substrates and catalytic groups on the enzyme optimally for conversion to the transition-state structure-- called orbital steering

12. Enzyme catalysis characterized by two features: specificity and rate acceleration
Active site contains amino acid residues and cofactors that are responsible for the above features
Cofactor, also called a coenzyme, is an organic molecule or metal ion that is essential for the catalytic action

13. Specificity of Enzyme-Catalyzed Reactions
Two types of specificity: (1) Specificity of binding and (2) specificity of reaction
Specificity of Binding
Enzyme catalysis is initiated by interaction between enzyme and substrate (ES complex)
k1, also referred to as kon, is rate constant for formation of the ES complex
k-1, also referred to as koff, is rate constant for breakdown of the complex
Stability of ES complex is related to affinity of the substrate for the enzyme as measured by Ks, dissociation constant for the ES complex

14. Generalized enzyme-catalyzed reaction
kon
Michaelis complex
koff
Scheme 1.1
When k2 << k-1,
k2 called kcat (turnover number)
Ks called Km (Michaelis-Menten constant)
kcat represents the maximum number of substrate molecules converted to product molecules per active site per unit of time; called turnover number

16. Km is the concentration of substrate that produces half the maximum rate
Km is a dissociation constant, so the smaller the Km the stronger the interaction between E and S
kcat/Km is the specificity constant - used to rank an enzyme according to how good it is with different substrates
kcat
Upper limit for is rate of diffusion (109 M-1s-1) Km

17. How does an enzyme release product so efficiently given that the enzyme binds the transition state structure about 1012 times more tightly than it binds the substrate or products?
After bond breaking (or making) at transition state, interactions that match in the transition-state stabilizing complex are no longer present.
Therefore products are poorly bound, resulting in expulsion.
As bonds are broken/made, changes in electronic distribution can occur, generating a repulsive interaction, leading to expulsion of products

18. E • S complex
Figure 1.1

19. ∆Gº = -RTlnKeq
If Keq = 0.01, ∆Gº of -5.5 kcal/mol needed to shift Keq to 100

20. Specific Forces Involved in E•S Complex Formation
Examples of ionic, ion-dipole, and dipole-dipole interactions. The wavy line represents the enzyme active site
Figure 1.2

21. Hydrogen bonding in the secondary structure of proteins: -helix and -sheet.
H-bonds
H-bonds
A type of dipole-dipole interaction between X-H and Y: (N, O)
Figure 1.3

22. Charge Transfer Complexes
When a molecule (or group) that is a good electron donor comes into contact with a molecule (or group) that is a good electron acceptor, donor may transfer some of its charge to the acceptor

23. Hydrophobic Interactions
When two nonpolar groups, each surrounded by water molecules, approach each other, the water molecules become disordered in an attempt to associate with the water molecules of the approaching group
Increases entropy, resulting in decrease in the free energy (G = H-TS)

24. van der Waals Forces
Atoms have a temporary nonsymmetrical distribution of electron density resulting in generation of a temporary dipole
Temporary dipoles of one molecule induce opposite dipoles in the approaching molecule

25. Binding Specificity Can be absolute or can be very broad
Specificity of racemates may involve E•S complex formation with only one enantiomer or E•S complex formation with both enantiomers, but only one is converted to product
Enzymes accomplish this because they are chiral molecules (mammalian enzymes consist of only L-amino acids)

26. Resolution of a racemic mixture
Binding specificity of enantiomers
Resolution of a racemic mixture
diastereomers
Scheme 1.2

27. Binding energy for E•S complex formation with one enantiomer may be much higher than that with the other enantiomer
Both E•S complexes may form, but only one E•S complex may lead to product formation
Enantiomer that does not turn over is said to undergo nonproductive binding

28. Basis for enantioselectivity in enzymes
Steric hindrance to binding of enantiomers
Basis for enantioselectivity in enzymes
Leu S R
Figure 1.4

29. Reaction Specificity
Unlike reactions in solution, enzymes can show specificity for chemically identical protons

30. Enzyme specificity for chemically identical protons
Enzyme specificity for chemically identical protons. R and R on the enzyme are groups that interact specifically with R and R, respectively, on the substrate.
Figure 1.5

31. Rate Acceleration
An enzyme has numerous opportunities to invoke catalysis:
Stabilization of the transition state
Destabilization of the E•S complex
Destabilization of intermediates
Because of these opportunities, multiple steps may be involved

32. Effect of (A) a chemical catalyst and (B) an enzyme on activation energy
Figure 1.6
fold typically

33. Enzyme catalysis does not alter the equilibrium of a reversible reaction; it accelerates attainment of the equilibrium

35. Mechanisms of Enzyme Catalysis Approximation
Rate enhancement by proximity
Enzyme serves as a template to bind the substrates
Reaction of enzyme-bound substrates becomes first order
Equivalent to increasing the concentration of the reacting groups
Exemplified with nonenzymatic model studies

36. Second-order reaction of acetate with aryl acetate
Scheme 1.3

37. Table 1.3. Effect of Approximation on Reaction Rates

38. Nucleophilic catalysis
Covalent Catalysis
Nucleophilic catalysis
anchimeric assistance
Scheme 1.4
Most common
Cys (SH)
Ser (OH)
His (imidazole)
Lys (NH2)
Asp/Glu (COO-)

39. Anchimeric assistance by a neighboring group
Scheme 1.5

40. Early evidence to support covalent catalysis
Model Reaction for Covalent Catalysis
Early evidence to support covalent catalysis
Scheme 1.6

41. General Acid/Base Catalysis
This is important for any reaction in which proton transfer occurs

42. The catalytic triad of -chymotrypsin
The catalytic triad of -chymotrypsin. The distances are as follows: d1 = 2.82 Å; d2 = 2.61 Å; d3 = 2.76 Å.
catalytic triad
Figure 1.7

43. Charge relay system for activation of an active-site serine residue in -chymotrypsin
Scheme 1.7

44. pKa values of amino acid side-chain groups within the active site of enzymes can be quite different from those in solution
Partly result of low polarity inside of proteins
Molecular dynamics simulations show interiors of these proteins have dielectric constants of about 2-3 (dielectric constant for benzene or dioxane)
If a carboxylic acid is in a nonpolar region, pKa will rise
Glutamate-35 in the lysozyme-glycolchitin complex has a pKa of 8.2; pKa in solution is 4.5
If the carboxylate ion forms salt bridge, it is stabilized and has a lower pKa

45. Basic group in a nonpolar environment has a lower pKa
pKa of a base will fall if adjacent to other bases
Active-site lysine in acetoacetate decarboxylase has a pKa of 5.9 (pKa in solution is 10.5)

46. Two kinds of acid/base catalysis:
Specific acid or specific base catalysis - catalysis by a hydronium (H3O+) or hydroxide (HO-) ion, and is determined only by the pH
General acid/base catalysis - reaction rate increases with increasing buffer concentration at a constant pH and ionic strength

47. Effect of the buffer concentration on (A) specific acid/base catalysis and (B) general acid/base catalysis
Specific acid/base catalysis
General acid/base catalysis
Figure 1.8

48. Hydrolysis of ethyl acetate
Specific Acid-Base Catalysis
Hydrolysis of ethyl acetate
Scheme 1.8

49. Alkaline hydrolysis of ethyl acetate
Scheme 1.9

50. Acid hydrolysis of ethyl acetate
Scheme 1.10

51. Simultaneous acid and base enzyme catalysis
Enzymes can utilize acid and base catalysis simultaneously
Simultaneous acid and base enzyme catalysis
acid catalysis
base catalysis
Scheme 1.11

52. Simultaneous acid/base catalysis is the reason for how enzymes are capable of deprotonating weak carbon acids

53. Simultaneous acid and base enzyme catalysis in the enolization of mandelic acid
Scheme 1.12

54. Low-barrier hydrogen bonds - short (< 2
Low-barrier hydrogen bonds - short (< 2.5Å), very strong hydrogen bonds
Stabilization of the enolic intermediate occurs via low-barrier hydrogen bonds

55. Simultaneous acid and base enzyme catalysis in the 1,4-elimination of -substituted (A) aldehydes, ketones, thioesters and (B) carboxylic acids
low-barrier H-bond
One-base mechanism
syn-elimination
“strong” acid
“weak” base
stronger acid needed
low-barrier H-bond
Two-base mechanism
anti-elimination
“strong” base
“weak” acid
Scheme 1.13
carboxylic acids

56. ElcB mechanism - not relevant
Base catalyzed 1,4-elimination of -substituted carbonyl compounds via an enolate intermediate (ElcB mechanism)
Needs acid or metal catalysis
Scheme 1.14

57. Electrostatic enzyme catalysis in enolization
Alternative to Low-Barrier Hydrogen Bond
Electrostatic enzyme catalysis in enolization
Scheme 1.15

58. Electrostatic stabilization of the transition state
Electrostatic Catalysis
Electrostatic stabilization of the transition state
oxyanion hole
Scheme 1.16

59. Desolvation The removal of water molecules at the active site
on substrate binding
Exposes substrate to lower dielectric constant environment
Exposes water-bonded charged groups for electrostatic catalysis
Destabilizes the ground state

60. Alkaline hydrolysis of phosphodiesters
Strain Energy
Alkaline hydrolysis of phosphodiesters
Scheme 1.17
k1.7
= 108
k1.8

61. Induced Fit Hypothesis putting strain energy into the substrate
Figure 1.9

62. Energetic Effect of Enzyme Catalysis
Figure 1.10
Importance of ground state destabilization

63. Mechanisms of Enzyme Catalysis - porphobilinogen synthase