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Enzyme Rate Enhancement

Enzyme Rate Enhancement. How do enzymes work to catalyze reactions?. Enzyme Classes by Reaction Type. Oxidation-Reduction Reaction: Oxidoreductase. Functional Group Transfer: Transferase. Cutting with Water: Hydrolysis. Group Elimination with Double Bond Formation: Lyase.

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Enzyme Rate Enhancement

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  1. Enzyme Rate Enhancement How do enzymes work to catalyze reactions?

  2. Enzyme Classes by Reaction Type

  3. Oxidation-Reduction Reaction: Oxidoreductase

  4. Functional Group Transfer: Transferase

  5. Cutting with Water: Hydrolysis

  6. Group Elimination with Double Bond Formation: Lyase

  7. Compound Conversion to an Isomeric Form: Isomerase Triose phosphate isomerase

  8. Bond Formation with ATP Hydrolysis: Ligase DNA Ligase -- nucleotide base coupling with loss of PPi

  9. Chymotrypsin Catalyzes Peptide Hydrolysis Enzyme specificity in terms of: Site of hydrolysis and Substrate reactivity How can a reaction be pushed in the forward direction?

  10. Reactions that are Spontaneous in the Forward and Reverse Direction How do enzymes affect the energy diagram of a reaction?

  11. Enzymes Decrease the Activation Energy How can enzymes lower the transition state? Is external energy required?

  12. Mechanisms of Enzyme Catalysis Acid-base, covalent, metal ion, orientation and proximity

  13. Ketone-Enol Conversion What mechanism of enzyme catalysis is operative here?

  14. Amino Acid Residues that Function in Acid-Base Catalysis

  15. Conventions for Depicting Reaction Mechanisms Curved arrow indicates electron rearrangement during a reaction

  16. Covalent Catalysis A formal positive charge favors electron flow to the nitrogen group

  17. Covalent Catalysis

  18. Amino Acids that can Participate in Covalent Catalysis

  19. Metal Ion Catalysis

  20. Components that Facilitate Enzyme Catalysis What is the cofactor in ATP hydrolysis? What is the co-substrate in an oxidation-reduction reaction?

  21. Enzymes Decrease the Activation Energy

  22. Mechanisms of Enzyme Catalysis • Acid-Base Catalysis • Covalent Catalysis • Metal Ion Catalysis • Orientation/Proximity Effects • Preferential Transition- State Binding

  23. Proximity and Orientation Effects Facilitate Catalysis

  24. Chymotrypsin Specificity Cleaves peptides on the C-terminus side of hydrophobic residues (e.g. Phe, Tyr and Try)

  25. Active Site Mapping via Irreversible Inhibitors Diisopropylphosphofluoridate (DIPF) inhibits chymotrypsin by modifying 1 of 28 serine residues

  26. Active Site Mapping via Irreversible Inhibitors

  27. Covalent Catalysis for Chymotrypsin: a Two Step Process • Enzyme acylation with leaving group departure • Enzyme deacylation What is the leaving group with Chymotrypsin?

  28. Chymotrypsin Catalysis Proceedsvia a Two-Step Mechanism Chromogenic substrate for kinetic studies Why is this compound not an ideal substrate mimic?

  29. Chymotrypsin Catalytic Triad • Catalytic triad serves as the site of catalysis • Aspartate and histidine contribute serine’s basicity • Serine serves as a nucleophile in covalent catalysis What type of catalysis occurs?

  30. Mechanism of Peptide Hydrolysis in Chymotrypsin Substrate binding via nucleophilic attack

  31. Mechanism of Peptide Hydrolysis in Chymotrypsin Polypeptide original C-side serves as leaving group

  32. Mechanism of Peptide Hydrolysis in Chymotrypsin Water attacks original N-side of polypeptide

  33. Mechanism of Peptide Hydrolysis in Chymotrypsin Polypeptide original N-side serves as leaving group and enzyme is regenerated

  34. Chymotrypsin Hydrolysis

  35. Tetrahedral-Intermediate Stabilization in Chymotrypsin H-bonds ideally positioned in the oxyanion hole stabilize the sp3 transition state

  36. Chymotrypsin Specificity Pocket Large structural pocket lined with hydrophobic amino acids favors bulky hydrophobic residues

  37. Serine Proteases Differ in Little Except Their Specificity Pockets ChymotrypsinTrypsinElastase

  38. Substrate Specificity Observed with each Proteolytic Enzyme • Papain cleave peptides non-selectively • Trypsin cleaves carboxyl side of bulky + charged R- groups • Chymotrypsin cleaves carboxyl side of bulky aromatic R-groups Thrombin

  39. Divergent Evolution Percent Sequence Identity among Three Serine Proteases Bovine trypsin 100% Bovine chymotrypsinogen 53% Porcine elatase 48% Common ancestor with retention of overall structure and catalytic mechanism

  40. Convergent Evolution Bovine versus bacterial serine protease No sequence or structural similarity but the same catalytic triad and oxyanion hole in the active site

  41. Enzyme-Substrate Binding Critical for Catalysis Lock and Key Model Enzyme Active Site • 3-D cleft or crevice • Small part of enzyme • Unique micro-environment • Substrate binding by weak forces Induced Fit Model

  42. Substrate-Induced Enzyme Conformational Change Substrate (+) Substrate (-)

  43. Inhibition by Transition State Analogs Pyrrolidine the natural substrate binds 160 less tightly than pyrrole a transition state analog. What is the favored enzyme binding geometry?

  44. Rate of Enzyme Catalysis Explain why enzyme activity increases with temperature and then precipitously drops off

  45. RNAas A Digestive Enzyme Cleaving Mechanism Why does ribonuclease catalyzes the hydrolysis of RNA but not DNA

  46. Conversion of Adenosine to Inosine What does the much greater binding affinity of 1,6-dihydropurine ribonucleoside than the substrate indicate about the enzyme mechanism?

  47. Chapter 6 Problems: 1, 3, 7, 9, 11, 15, 20, 23, 25 and 37

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