Mechanism of Enzymes

1. Mechanism of Enzymes Mechanisms the molecular details of catalyzed reactions

2. 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

3. Two types of nucleophilic substitution reactions • Direct displacement

4. Two types of nucleophilic substitution reactions • Formation of a tetrahedral intermediate

5. Cleavage reactions • Carbanion formation • Carbocation formation

6. Cleavage reactions • Free radical formation

7. Catalysts Stabilize Transition States Energy diagram for a single-step reaction

8. 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

9. Enzymes lower the activation energy of a reaction • (1) Substratebinding • Enzymes properly position substrates for reaction(makes the formation of the transition state more frequent and lowers the energy of activation) • (2) Transitionstatebinding • Transition states are bound more tightly than substrates (this also lowers the activation energy)

10. Enzymatic catalysis of the reaction A+B A-B

11. 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

12. Acid-Base Catalysis • Reaction acceleration is achieved by catalytictransferofaproton • A general base (B:) can act as a protonacceptor to remove protons from OH, NH, CH or other XH • This produces a stronger nucleophilic reactant (X:-)

13. 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

14. 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)

15. Covalent Catalysis Stepone: a glucosyl residue is transferred to enzyme *Sucrose + Enz Glucosyl-Enz + Fructose Steptwo: Glucose is donated to phosphate Glucosyl-Enz + PiGlucose 1-phosphate + Enz *(Sucrose is composed of a glucose and a fructose)

16. Triose Phosphate Isomerase (TPI) • TPI catalyzes a rapid aldehyde-ketone interconversion

17. Proposed mechanism for TPI • General acid-base catalysis mechanism (4 slides)

18. Proposed mechanism for TPI

19. Proposed mechanism for TPI

20. Proposed mechanism for TPI

21. Energy diagram for the TPI reaction

22. Binding Modes of Enzymatic Catalysis • Proper binding of reactants in enzyme active sites provides substratespecificity and catalyticpower • 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

23. Binding forces utilized for catalysis 1. Charge-charge interactions 2. Hydrogen bonds 3. Hydrophobic interactions 4. Van der Waals forces

24. The Proximity Effect • Correctpositioning 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

25. Reactions of carboxylates with phenyl esters

26. Reactions of carboxylates with phenyl esters

27. Reactions of carboxylates with phenyl esters

28. 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

29. 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)

30. 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

31. Transition-state (TS) analogs • Transition-state analogs are stable compounds whose structures resemble unstable transition states • 2-Phosphoglycolate, a TS analog for the enzyme triose phosphate isomerase

32. Glucose + ATP Glucose 6-phosphate + ADP 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

33. 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

34. 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)

35. Conformations of N-acetylmuramic acid (a) Chair conformation (b) D-Site sugar residue is distorted into a higher energy half-chair conformation

36. Lysozyme reaction mechanisms 1. Proximity effects 2. Acid-base catalysis 3. TS stabilization (or substrate distortion toward the transition state)

37. Mechanism of lysozyme

39. Properties of Serine Proteases 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

40. Activation of some pancreatic zymogens

41. 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

42. Binding sites of chymotrypsin, trypsin, and elastase • Substrate specificities are due to relatively small structural differences in active-site binding cavities

43. Identification of His at active site • The irreversible inhibitor (TosPheCH2Cl) binds to the active-site His residue in serine proteases

44. 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

45. a-Chymotrypsin mechanism

49. (Acyl E + H2O)

50. (E-TI2)