Chapter 14 Mechanisms of Enzyme Action

1. Chapter 14Mechanisms 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. 14.3.

6. 14.2 What Role Does Transition-State Stabilization Play in Enzyme Catalysis? Figure 14.1 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. 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 14.2 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 10 -20 to 10-26 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 14.5 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 430-431).

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 0.0001% 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 14.9 Human cyclophilin A is a prolyl isomerase, which catalyzes the interconversion between transand cis conformations of proline in peptides.

33. Protein Motions Are Essential to Enzyme Catalysis Figure 14.9 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 14.11 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 14.11 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 14.11 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 14.12.

40. General Acid-base Catalysis Figure 14.12 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 10-30 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 14.13 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 14.14 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. 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.