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. 14.2 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 X‡ • 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

5. 14.2 What Role Does Transition-State Stabilization Play in Enzyme Catalysis? 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.

6. 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 cannot be "too tight" - goal is to make the energy barrier between ES and EX‡ small

7. 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 • distortion • desolvation

8. 14.3 How Does Destabilization of ES Affect Enzyme Catalysis? (a) Catalysis does not occur if ES and X‡ are equally stabilized. (b) Catalysis will occur if X‡ is stabilized more than ES.

9. 14.3 How Does Destabilization of ES Affect Enzyme Catalysis? (a) Formation of the ES complex results in entropy loss. The ES complex is a more highly ordered, low-entropy state for the substrate.

10. 14.3 How Does Destabilization of ES Affect Enzyme Catalysis? (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.

11. 14.3 How Does Destabilization of ES Affect Enzyme Catalysis? (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.

12. 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 10-26M! • 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

13. 14.4 How Tightly Do Transition-State Analogs Bind to the Active Site? The proline racemase reaction. Pyrrole-2-carboxylate and Δ-1-pyrroline-2-carboxylate mimic the planar transition state of the reaction.

14. 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 lowers blood pressure • Statins lower serum cholesterol • Protease inhibitors are AIDS drugs • Juvenile hormone esterase is a pesticide target • Tamiflu is a viral neuraminidase inhibitor

15. How many other drug targets might there be? • The human genome contains approximately 20,000 genes • How many might be targets for drug therapy? • More than 3000 experimental drugs are presently under study and testing • These and many future drugs will be designed as transition-state analog inhibitors • See the DrugBank: http://drugbank.ca

16. 14.5 What Are the Mechanisms of Catalysis? • Enzymes facilitate formation of near-attack complexes • Protein motions are essential to enzyme catalysis • Covalent catalysis • General acid-base catalysis • Low-barrier hydrogen bonds • Metal ion catalysis

17. 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) • NACs are precursors to reaction transition states

18. 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

19. 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.

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

21. 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

22. 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

23. Covalent Catalysis Examples of covalent enzyme-substrate intermediates.

24. Covalent Catalysis

25. 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

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

27. 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

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

29. Quantum Mechanical Tunneling • Tunneling provides a path “around” the usual energy of activation for steps in chemical reactions • Many enzymes exploit this • According to quantum theory, there is a finite probability that any particle can appear on the other side of an activation barrier for a reaction step • The likelihood of tunneling depends on the distance over which a particle must move • Only protons and electrons have a significant probability of tunneling

30. Quantum Mechanical Tunneling • The de Broglie equation relates the “de Broglie wavelength” to the mass and energy of a particle • Tunneling can only play a significant role in a reaction when the wavelength of the transferring particle is similar to the distance over which it is transferred • de Broglie wavelengths for protons and electrons are 0.9Å and 38Å, respectively • Tunneling probably contributes to most, if not all, hydrogen transfer reactions

31. Tunneling between donor and acceptor If the distance for particle transfer is sufficiently small, overlap of probability functions (red) permit efficient quantum mechanical tunneling between donor (D) and acceptor (A)

32. Metal Ion Catalysis 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.

33. 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

34. 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

35. 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

36. The Catalytic Triad of the Serine Proteases 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.

37. The Catalytic Triad of the Serine Proteases The catalytic triad at the active site of chymotrypsin (and the other serine proteases.)

38. Serine Protease Binding Pockets are Adapted to Particular Substrates 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.

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

40. 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 backbone N-H groups of Gly193 and Ser195

41. The Serine Protease Mechanism in Detail Figure 14.21 The chymotrypsin mechanism: binding of a model substrate.

42. The Serine Protease Mechanism in Detail Figure 14.21 The chymotrypsin mechanism: the formation of the covalent ES complex (E-Ser195–S complex) involves general base catalysis by His57

43. The Serine Protease Mechanism in Detail Figure 14.21 The chymotrypsin mechanism: His57 stabilized by a LBHB.

44. The Serine Protease Mechanism in Detail Figure 14.21 The chymotrypsin mechanism: collapse of the tetrahedral intermediate releases the first product.

45. The Serine Protease Mechanism in Detail Figure 14.21 The chymotrypsin mechanism: The amino product departs, making room for an entering water molecule.

46. The Serine Protease Mechanism in Detail Figure 14.21 The chymotrypsin mechanism: Nucleophilic attack by water is facilitated by His57, acting as a general base.

47. The Serine Protease Mechanism in Detail Figure 14.21 The chymotrypsin mechanism: Collapse of the tetrahedral intermediate cleaves the covalent intermediate, releasing the second product.

48. The Serine Protease Mechanism in Detail Figure 14.21 The chymotrypsin mechanism: Carboxyl product release completes the serine protease mechanism.

49. The Serine Protease Mechanism in Detail Figure 14.21 The chymotrypsin mechanism: At the completion of the reaction, the side chains of the catalytic triad are restored to their original states.

50. Transition-State Stabilization in the Serine Proteases • The chymotrypsin mechanism involves two tetrahedral oxyanion transition states • These transition states are stabilized by a pair of amide groups that is termed the “oxyanion hole” • The amide N-H groups of Ser195 and Gly193 provide primary stabilization of the tetrahedral oxyanion