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Catalytic Mechanisms

Catalytic Mechanisms. Objective. To understand how enzymes work at the molecular level. Ultimately requires total structure determination, but can learn much through biochemical analysis. To Be Explained. Specificity For specific substrates Amino acids residues involved Catalysis

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Catalytic Mechanisms

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  1. Catalytic Mechanisms

  2. Objective To understand how enzymes work at the molecular level. Ultimately requires total structure determination, but can learn much through biochemical analysis.

  3. To Be Explained • Specificity • For specific substrates • Amino acids residues involved • Catalysis • Mechanisms • Amino acids involved/Specific role(s)

  4. Enzyme Binding Sites • Active Site: • Substrate Binding Site + Catalytic Site • Regulatory Site: • a second binding site, • Binding by regulatory molecule affects the active site • alter the efficiency of catalysis • improve or inhibit

  5. General Characteristics • Three dimensional space • Occupies small part of enzyme volume • Clefts or crevices • Ligands (substrate or effector) bound by multiple weak interactions • Specificity depends on precise arrangement of atoms in active site

  6. Models Induced Fit Lock and Key

  7. Identification and Characterization of Active Site • Structure: size, shape, charges, etc. • Composition: identify amino acids involved in binding and catalysis.

  8. Binding or Positioning Site(Trypsin)

  9. Binding or Positioning Site(Chymotrypsin)

  10. Catalytic Site(e.g. Chymotrypsin)

  11. Probing the Structure of the Active Site Model Substrates

  12. Model Substrates(Chymotrypsin)

  13. Peptide Chain? All Good Substrates!

  14. a-amino group? Good Substrate!

  15. Side Chain Substitutions Good Substrates t-butyl- Cyclohexyl

  16. ConclusionBulky Hydrophobic Binding Site

  17. Probing the Structure of the Active Site Competitive Inhibitors

  18. Arginase

  19. Good Competitive Inhibitors

  20. Poor Competitive Inhibitors All Three Charged Groups are Important

  21. ConclusionActive Site Structure of Arginase

  22. Identifying Active Site Amino Acid Residues • Covalent modification of residues • Inactivation of enzyme • Site directed mutagenesis • Inactivation of enzyme

  23. Mechanisms of Catalysis • Acid-base catalysis • Covalent catalysis • Metal ion catalysis • Proximity and orientation effects • Preferential binding (stabilization) of the transition state

  24. Acid-Base Catalysis Addition or removal of a proton by side chains

  25. General Acids and Bases

  26. Acid-Base Catalysis Keto-EnolTautomerization

  27. Uncatalyzed Reaction

  28. General Acid Catalysis

  29. General Base Catalysis

  30. Ribonuclease A Figure 11-10

  31. Mechanism of RNase A Figure 11-10 part 1

  32. Mechanism of RNase A Figure 11-10 part 2

  33. Covalent Catalysis(Nucleophilic catalysis)(Principle) Involves a transient covalent bond between the enzyme and the substrate Usually by the nucleophilic attack of the substrate by the enzyme

  34. Covalent Catalysis(Principle) Slow H2O + A–B ——> AOH + BH A-B + E-H ——> E-A + BH E-A + H2O ——> A-OH + E-H Fast NOTE: New Reaction Pathway

  35. Covelent Catalysis

  36. The Schiff Base

  37. Metal Ion Catalysis • Charge stabilization • Water ionization • Charge shielding

  38. Metal Ion Catalysis • Metalloenzymes: tightly bound metal ions • Catalytically essential • Fe2+, Fe3+, Cu2+, Mn2+, and Co2+ • Metal-activated enzymes: loosely bound metal ions (from solution or with substrate) • Structural metal ions: • Na+, K+, and Ca2+ • Both: Mg2+ and Zn2+

  39. Carbonic Anhydrase

  40. Carbonic Anhydrase

  41. Proximity and Orientation EffectsRate of a reaction depends on: • Number of collisions • Energy of molecules • Orientation of molecules • Reaction pathway (transition state)

  42. Proximity V = k[A][B] [A] and [B] = ~13M on enzyme surface

  43. Biomolecular Reaction of Imidazole with p-Nitrophenylacetate(Intermolecular) Page 336

  44. Intramolecular Reaction of Imidazole with p-Nitrophenylacetate(Intramolecular) Intramolecular Rate = 24x Intermolecular Rate Page 336

  45. Orientation

  46. Geometry of an SN2 Reaction Figure 11-14

  47. Preferrential Binding of Reaction Intermediate • Stabilize Transition State • Electrostatic stabilization of developing charge • Relief of induced bond angle strain • Enhancement of weak interactions between enzyme and intermediate.

  48. Steric Strain in Organic Reactions Reaction Rate: R=CH3 is 315x vs R=H Page 338

  49. Effect of PreferentialTransition State Binding Figure 11-15

  50. Transition State Analogs Powerful Enzyme Inhibitors

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