1. Chapter 14 Mechanisms of Enzyme Action

2. Outline What are the magnitudes of enzyme-induced rate accelerations ?
What role does transition-state stabilization play in enzyme catalysis ?
How does destabilization of ES affect enzyme catalysis ?
How tightly do transition-state analogs bind to the active site ?
What are the mechanisms of catalysis ?
What can be learned from typical enzyme mechanisms ?

3. 14.1 What Are the Magnitudes of Enzyme-Induced Rate Accelerations?
Enzymes are powerful catalysts.
The large rate accelerations of enzymes (107 to 1015 correspond to large changes in the free energy of activation for the reaction.
All reactions pass through a transition state on the reaction pathway.
The active sites of enzymes bind the transition state of the reaction more tightly than the substrate.
By doing so, enzymes stabilize the transition state and lower the activation energy of the reaction.

4. 14.1 What Are the Magnitudes of Enzyme-Induced Rate Accelerations?

5. 14.2 What Role Does Transition-State Stabilization Play in Enzyme Catalysis?
The catalytic role of an enzyme is to reduce the energy barrier between substrate S and transition state.
Rate acceleration by an enzyme means that the energy barrier between ES and EX‡ must be smaller than the barrier between S and X‡.
This means that the enzyme must stabilize the EX‡ transition state more than it stabilizes ES.
See Eq

6. 14.2 What Role Does Transition-State Stabilization Play in Enzyme Catalysis?
Figure Enzymes catalyze reactions by lowering the activation energy. Here the free energy of activation for (a) the uncatalyzed reaction is larger than that of the enzyme-catalyzed reaction.

7. Competing effects determine the position of ES on the energy scale
14.2 What Role Does Transition-State Stabilization Play in Enzyme Catalysis?
Competing effects determine the position of ES on the energy scale
Try to mentally decompose the binding effects at the active site into favorable and unfavorable.
The binding of S to E must be favorable.
But not too favorable!
Km should not be "too tight" - goal is to make the energy barrier between ES and EX‡ small.

8. 14.3 How Does Destabilization of ES Affect Enzyme Catalysis?
Raising the energy of ES raises the rate
For a given energy of EX‡, raising the energy of ES will increase the catalyzed rate.
This is accomplished by:
a) loss of entropy due to formation of ES.
b) destabilization of ES by:
Strain or distortion.
Desolvation.
Electrostatic effects.

9. 14.3 How Does Destabilization of ES Affect Enzyme Catalysis?
Figure The intrinsic binding energy of ES is compensated by entropy loss due to binding of E and S and by destabilization due to strain and distortion.

10. 14.3 How Does Destabilization of ES Affect Enzyme Catalysis?
Figure 14.3(a) Catalysis does not occur if ES and X‡ are equally stabilized. (b) Catalysis will occur if X‡ is stabilized more than ES.

11. 14.3 How Does Destabilization of ES Affect Enzyme Catalysis?
Figure 14.4(a) Formation of the ES complex results in entropy loss. The ES complex is a more highly ordered, low-entropy state for the substrate.

12. 14.3 How Does Destabilization of ES Affect Enzyme Catalysis?
Figure 14.4(b) Substrates typically lose waters of hydration in the formation in the formation of the ES complex. Desolvation raises the energy of the ES complex, making it more reactive.

13. 14.3 How Does Destabilization of ES Affect Enzyme Catalysis?
Figure 14.4(c) Electrostatic destabilization of a substrate may arise from juxtaposition of like charges in the active site. If charge repulsion is relieved in the reaction, electrostatic destabilization can result in a rate increase.

14. 14.4 How Tightly Do Transition-State Analogs Bind to the Active Site?
Very tight binding to the active site
The affinity of the enzyme for the transition state may be to M!
Can we see anything like that with stable molecules ?
Transition state analogs (TSAs) are stable molecules that are chemically and structurally similar to the transition state.
Proline racemase was the first case.
See Figure 14.6 for some recent cases.

15. 14.4 How Tightly Do Transition-State Analogs Bind to the Active Site?
Figure The proline racemase reaction.
Pyrrole-2-carboxylate and Δ-1-pyrroline-2-carboxylate mimic the planar transition state of the reaction.

16. 14.4 How Tightly Do Transition-State Analogs Bind to the Active Site?
Figure 14.6(a) Phosphoglycolohydroxamate is an analog of the enediolate transition state of the yeast aldolase reaction.

17. 14.4 How Tightly Do Transition-State Analogs Bind to the Active Site?
(b) Purine riboside inhibits adenosine deaminase.
The hydrated form is an analog of the transition state of the reaction.

18. Transition-State Analogs Make Our World Better
Enzymes are often targets for drugs and other beneficial agents.
Transition state analogs often make ideal enzyme inhibitors.
Enalapril and Aliskiren lower blood pressure.
Statins lower serum cholesterol.
Protease inhibitors are AIDS drugs.
Juvenile hormone esterase is a pesticide target.
Tamiflu is a viral neuraminidase inhibitor.

19. How to read and write mechanisms
The custom of writing chemical reaction mechanisms with electron dots and curved arrows began with Gilbert Newton Lewis and Sir Robert Robinson.
Review of Lewis dot structures, the concepts of valence electrons and formal charge.
Formal charge = group number – nonbonding electrons – (1/2 shared electrons).
Electronegativity is also important:
F > O > N > C > H.
The arrows used always point from where electrons originate to where they are going.

20. How to read and write mechanisms
For a bond-breaking event, the arrow begins in the middle of the bond, and the arrow points to the atom that will accept the electrons.
For a bond-making event, the arrow begins at the source of the electrons (for example, a nonbonded pair), and the arrowhead points to the atom where the new bond will be formed.

21. How to read and write mechanisms
It has been estimated that 75% of the steps in enzyme reaction mechanisms are proton (H+) transfers.
If the proton is donated or accepted by a group on the enzyme, it is often convenient (and traditional) to represent the group as “B”, for “base”, even if B is protonated and behaving as an acid:

22. How to read and write mechanisms
It is important to appreciate that a proton transfer can change a nucleophile into an electrophile, and vice versa.
Thus, it is necessary to consider:
The protonation states of substrate and active-site residues (mostly the sidechains).
How pKa values of sidechains can change in the environment of the active site.
For example, an active-site histidine, which might normally be protonated, can be deprotonated by another group and then act as a base, accepting a proton from the substrate.

23. How to read and write mechanisms
An active-site histidine, which might normally be protonated, can be deprotonated by another group and then act as a base, accepting a proton from the substrate.

24. How to read and write mechanisms
Water can often act as an acid or base at the active site through proton transfer with an assisting active-site residue:
This type of chemistry is the basis for general acid-base catalysis (discussed on pages ).

25. 14.5 What Are the Mechanisms of Catalysis?
Enzymes facilitate formation of near-attack complexes (NAC).
Protein motions are essential to enzyme catalysis.
Covalent catalysis.
General acid-base catalysis.
Low-barrier hydrogen bonds.
Metal ion catalysis.

26. Enzymes facilitate formation of near-attack complexes
X-ray crystal structure studies and computer modeling have shown that the reacting atoms and catalytic groups are precisely positioned for their roles.
Such preorganization selects substrate conformations in which the reacting atoms are in van der Waals contact and at an angle resembling the bond to be formed in the transition state.
Thomas Bruice has termed such arrangements near-attack conformations (NACs).

27. Enzymes facilitate formation of near-attack complexes
Thomas Bruice has proposed that near-attack conformations are precursors to transition states.
In the absence of an enzyme, potential reactant molecules adopt a NAC only about % of the time.
On the other hand, NACs have been shown to form in enzyme active sites from 1% to 70% of the time.

28. Enzymes facilitate formation of near-attack complexes
Figure 14.7
NACs are characterized as having reacting atoms within 3.2 Å and an approach angle of ±15° of the bonding angle in the transition state.

29. Enzymes facilitate formation of near-attack complexes
Figure 14.7
In an enzyme active site, the NAC forms more readily than in the uncatalyzed reaction. The energy separation between the NAC and the transition state is approximately the same in the presence and absence of the enzyme.

30. Figure 14.8 The active site of liver alcohol dehydrogenase – a near-attack complex.

31. Protein Motions Are Essential to Enzyme Catalysis
Proteins are constantly moving – bonds vibrate, side chains bend and rotate, backbone loops wiggle and sway, and whole domains move as a unit.
Enzymes depend on such motions to provoke and direct catalytic events.
Protein motions support catalysis in several ways. Active site conformation changes can:
Assist substrate binding.
Bring catalytic groups into position.
Induce formation of NACs.
Assist in bond making and bond breaking.
Facilitate conversion of substrate to product.

32. Protein Motions Are Essential to Enzyme Catalysis
Figure Human cyclophilin A is a prolyl isomerase, which catalyzes the interconversion between trans and cis conformations of proline in peptides.

33. Protein Motions Are Essential to Enzyme Catalysis
Figure The active site of cyclophilin with a bound peptide containing proline in cis and trans conformations. Motion by active site residues promote catalysis in cyclophilin.

34. Covalent Catalysis
Some enzymes derive much of their rate acceleration from formation of covalent bonds between enzyme and substrate.
The side chains of amino acids in proteins offer a variety of nucleophilic centers for catalysis.
These groups readily attack electrophilic centers of substrates, forming covalent enzyme-substrate complexes.
The covalent intermediate can be attacked in a second step by water or by a second substrate, forming the desired product.

35. Covalent Catalysis
Figure Examples of covalent bond formation between enzyme and substrate. A nucleophilic center X: on an enzyme attacks a phosphorus atom to form a phosphoryl enzyme intermediate.

36. Covalent Catalysis
Figure Examples of covalent bond formation between enzyme and substrate. A nucleophilic center X: on an enzyme attacks a carbonyl C to form an acyl enyzme intermediate.

37. Covalent Catalysis
Figure Examples of covalent bond formation between enzyme and substrate. A nucleophilic center X: attacks the anomeric carbon of a glycoside, forming a glucosyl enzyme intermediate.

38. Covalent Catalysis

39. General Acid-base Catalysis
Catalysis in which a proton is transferred in the transition state
"Specific" acid-base catalysis involves H+ or OH- that diffuses into the catalytic center.
"General" acid-base catalysis involves acids and bases other than H+ and OH-.
These other acids and bases facilitate transfer of H+ in the transition state.
See Figure

40. General Acid-base Catalysis
Figure Catalysis of p-nitrophenylacetate hydrolysis can occur either by specific acid hydrolysis or by general base catalysis.

41. Low-Barrier Hydrogen Bonds (LBHBs)
The typical H-bond strength is kJ/mol, and the O-O separation is typically 0.28 nm.
As distance between heteroatoms becomes smaller (<0.25 nm), H bonds become stronger.
Stabilization energies can approach 60 kJ/mol in solution.
pKa values of the two electronegative atoms must be similar.
Energy released in forming an LBHB can assist catalysis.

42. Low-Barrier Hydrogen Bonds (LBHBs)
Figure Energy diagrams for conventional H bonds (a), and low-barrier hydrogen bonds (b and c).s In (c), the O-O distance is 0.23 to 0.24 nm, and bond order for each O-H interaction is 0.5.

43. Metal Ion Catalysis
Figure Thermolysin is an endoprotease with a catalytic Zn2+ ion in the active site. The Zn2+ ion stabilizes the buildup of negative charge on the peptide carbonyl oxygen, as a glutamate residue deprotonates water, promoting hydroxide attack on the carbonyl carbon.

44. How Do Active-Site Residues Interact to Support Catalysis?
About half of the amino acids engage directly in catalytic effects in enzyme active sites.
Other residues may function in secondary roles in the active site:
Raising or lowering catalytic residue pKa values.
Orientation of catalytic residues.
Charge stabilization.
Proton transfers via hydrogen tunneling.

45. How Do Active-Site Residues Interact to Support Catalysis?
The active site of aromatic amine dehydrogenase, showing the relationship of Asp128, Thr172, and Cys171. Coupling of local motions of these residues to vibrational states involved in proton transfer contributes to catalysis.

46. 14.5 What Can Be Learned From Typical Enzyme Mechanisms?
First Example: the serine proteases
Enzyme and substrate become linked in a covalent bond at one or more points in the reaction pathway.
The formation of the covalent bond provides chemistry that speeds the reaction.
Serine proteases also employ general acid-base catalysis.

47. Trypsin, chymotrypsin, elastase, thrombin, subtilisin, plasmin, TPA
The Serine Proteases
Trypsin, chymotrypsin, elastase, thrombin, subtilisin, plasmin, TPA
All involve a serine in catalysis - thus the name.
Ser is part of a "catalytic triad" of Ser, His, Asp.
Serine proteases are homologous, but locations of the three crucial residues differ somewhat.
Enzymologists agree, however, to number them always as His57, Asp102, Ser195.
Burst kinetics yield a hint of how they work.

48. The Serine Proteases Figure 14.15
The amino acid sequences of chymotrypsinogen, trypsin, and elastase.

49. The Catalytic Triad of the Serine Proteases
Figure 14.16
Structure of chymotrypsin (white) in a complex with eglin C (blue ribbon structure), a target substrate. His57 (red) is flanked by Asp102 (gold) and Ser195 (green). The catalytic site is filled by a peptide segment of eglin. Note how close Ser195 is to the peptide that would be cleaved in the reaction.

50. The Catalytic Triad of the Serine Proteases
Figure 14.17
The catalytic triad at the active site of chymotrypsin (and the other serine proteases.

51. Serine Protease Binding Pockets are Adapted to Particular Substrates
Figure The substrate-binding pockets of trypsin, chymotrypsin, and elastase. Asp189 (aqua) coordinates Arg and Lys residues of substrates in the trypsin pocket. Val216 (purple) and Thr226 (green) make the elastase pocket shallow and able to accommodate only small, nonbulky residues.
The chymotrypsin pocket is hydrophobic.

52. Serine Proteases Cleave Simple Organic Esters, such as p-Nitrophenylacetate
Figure Chymotrypsin cleaves simple esters, in addition to peptide bonds. P-Nitrophenylacetate has been used in studies of the chymotrypsin mechanism.

53. Serine Proteases Display Burst Kinetics
Figure 14.20
Burst kinetics in the chymotrypsin reaction.
p-NO2-phenol
Absorbs at 400 nm.
Covalent catalysis 
Acyl enzyme

54. Serine Protease Mechanism
A mixture of covalent and general acid-base catalysis
Asp102 functions only to orient His57.
His57 acts as a general acid and base.
Ser195 forms a covalent bond with peptide to be cleaved.
Covalent bond formation turns a trigonal C into a tetrahedral C.
The tetrahedral oxyanion intermediate is stabilized by the peptide N-H of Gly193 and of Ser195 in what is referred to as an “oxyanion hole”.

55. The Serine Protease Mechanism in Detail
Figure The chymotrypsin mechanism: binding of a model substrate.

56. The Serine Protease Mechanism in Detail
Figure The chymotrypsin mechanism: the formation of the covalent ES complex involves general base catalysis by His57.

57. The Serine Protease Mechanism in Detail
oxyanion
Figure The chymotrypsin mechanism: His57 stabilized by a LBHB. The tetrahedral intermediate in this diagram is an oxyanion.

58. The Serine Protease Mechanism in Detail
oxyanion
Figure The chymotrypsin mechanism: collapse of the tetrahedral intermediate releases the first product.

59. The Serine Protease Mechanism in Detail
Figure The chymotrypsin mechanism: The amino product departs, leaving the acylated enzyme and making room for an entering water molecule.

60. The Serine Protease Mechanism in Detail
Figure The chymotrypsin mechanism: Nucleophilic attack by water is facilitated by His57, acting as a general base.

61. The Serine Protease Mechanism in Detail
oxyanion
Figure The chymotrypsin mechanism: A second oxyanion from attack by water. Collapse of the tetrahedral intermediate cleaves the covalent intermediate, releasing the second product.

62. The Serine Protease Mechanism in Detail
Figure The chymotrypsin mechanism: Carboxyl product release completes the serine protease mechanism.

63. The Serine Protease Mechanism in Detail
Figure The chymotrypsin mechanism: At the completion of the reaction, the side chains of the catalytic triad are restored to their original states.

64. Transition-State Stabilization in the Serine Proteases
The chymotrypsin mechanism involves two tetrahedral oxyanion intermediates.
These intermediates are stabilized by a pair of amide groups in the “oxyanion hole”.
The peptide (amide) N-H groups that form this oxyanion hole and provide primary stabilization of the tetrahedral intermediates are from of Ser195 and Gly193.

65. The “oxyanion hole”
The oxyanion hole of chymotrypsin stabilizes the tetrahedral oxyanion intermediate seen in the mechanism of Figure

66. Aspartic proteases play many roles in humans

67. The Aspartic Proteases
Pepsin, chymosin, cathepsin D, renin and HIV-1 protease
All involve two Asp residues at the active site.
These two Asp residues work together as general acid-base catalysts.
Most aspartic proteases have a tertiary structure consisting of two lobes (N-terminal and C-terminal) with approximate two-fold symmetry.
HIV-1 protease is a homodimer.

68. The Aspartic Proteases
Most aspartic proteases exhibit a two-lobed structure. Each lobe contributes one catalytic aspartate to the active site. HIV-1 protease is a homodimeric enzyme, with each subunit contributing a catalytic Asp residue.
Figure 14.22
Structures of (a) HIV-1 protease, a dimer, and (b) pepsin, a monomer. Pepsin’s N-terminal half is shown in red; the C-terminal half is shown in blue.

69. The Aspartic Proteases
Figure 14.23
pH-rate profile for pepsin.

70. The Aspartic Proteases
Figure 14.23
pH-rate profile of HIV-1 protease.

71. Aspartic Protease Mechanism
Aspartic proteases show one relatively low pKa, and one relatively high pKa.
These pKa values as determined from the pH-rate plot for the enzyme, have traditionally been attributed to the two aspartate residues.
However, molecular dynamics simulations now show that aspartic proteases employ low-barrier hydrogen bonds (LBHBs) in their mechanism.
The predominant catalytic factor in aspartic proteases is general acid-base catalysis.

72. A Mechanism for the Aspartic Proteases
Figure Mechanism for the aspartic proteases.
LBHBs play a role in states E, ES, ET’, EQ’, and EP’Q.

73. A novel aspartic protease
HIV-1 Protease
A novel aspartic protease
HIV-1 protease cleaves the polyprotein products of the HIV genome.
This is a remarkable imitation of mammalian aspartic proteases.
HIV-1 protease is a homodimer - more genetically economical for the virus.
Active site is two-fold symmetric.

74. Proteolytic cleavage pattern for the HIV genome
Figure HIV mRNA provides the genetic information for synthesis of a polyprotein. Cleavage yields the active products.

75. Protease Inhibitors Block the Active Site of HIV-1 Protease
Figure HIV-1 protease complexed with the inhibitor Crixivan (red) made by Merck. The “flaps” that cover the active site are green; the catalytic active site Asp residues are violet.

76. Protease Inhibitors Give Life to AIDS Patients
Protease inhibitors as AIDS drugs
If the HIV-1 protease can be selectively inhibited, then new HIV particles cannot form.
Several novel protease inhibitors are currently marketed as AIDS drugs.
Many such inhibitors work in a culture dish.
However, a successful drug must be able to kill the virus in a human subject without blocking other essential proteases in the body.

77. End Chapter 14 Mechanisms of Enzyme Action