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Chapter 8: Mechanisms and Inhibitors

Chapter 8: Mechanisms and Inhibitors. What type of protein modifications might occur due to short- and long-term temperature changes?. Temperature Alters Enzymatic Activity. Tyrosinase Activity Curve. Tyrosinase Pigment synthesis enzyme Heat sensitive.

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Chapter 8: Mechanisms and Inhibitors

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  1. Chapter 8: Mechanisms and Inhibitors What type of protein modifications might occur due to short- and long-term temperature changes?

  2. Temperature Alters Enzymatic Activity Tyrosinase Activity Curve • Tyrosinase • Pigment synthesis enzyme • Heat sensitive Why are Siamese markings only on their extremities?

  3. [H+] Regulates Enzymatic Activity • Protein Digestive Enzymes • Pepsin stomach • Chymotrypsin • intestine Why do enzymes have a relatively narrow pH optimum?

  4. Competitive Enzyme Inhibition Reaction Pathway Is Vmax affected? How is KM influenced?

  5. Competitive Enzyme Inhibition Reaction Pathway Substrate can out compete inhibitor → Vmax unchanged since Vmax= k2[E]T Inhibitor binds in the active site → KM increases since KM= (k-1 + k2)/k1 How is Lineweaver-Burk plot altered?

  6. Competitive Inhibition – Lineweaver-Burk Plot • Vmax Unaltered • KM Increased

  7. Noncompetitive Enzyme Inhibition Reaction Pathway Is Vmax affected? How is KM influenced?

  8. Noncompetitive Enzyme Inhibition Reaction Pathway Taking enzyme out of circulation → Vmax lowered sinceVmax= k2[E]T Inhibitor binds both E and ES → KM unchanged since KM= (k-1 + k2)/k1 How is Lineweaver-Burk plot altered?

  9. Noncompetitive Inhibition – Lineweaver-Burk Plot • Vmax Lowered • KM Unchanged

  10. Uncompetitive Enzyme Inhibition Reaction Pathway Is Vmax affected? How is KM influenced?

  11. Uncompetitive Enzyme Inhibition Reaction Pathway Taking enzyme out of circulation → Vmax lowered since Vmax= k2[E]T Inhibitor binds both E and ES → KM decreasedsince KM= (k-1 + k2)/k1 How is Lineweaver-Burk plot altered?

  12. Uncompetitive Inhibition – Lineweaver-Burk Plot • Vmax Lowered • KM Lowered

  13. Inhibitor Identification • No Inhibition? • Competitive Inhibition? • Uncompetitive • Inhibition? • Noncompetitive • Inhibition?

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

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

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

  17. 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?

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

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

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

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

  22. Chymotrypsin Hydrolysis

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

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

  25. Active Site Mapping via Irreversible Inhibitors

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

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

  28. Cleavage of this amide substrate by chymotrypsin does not exhibit a two phase burst and steady state kinetic profile; what conclusions can be drawn? Product

  29. Sugar-Peptidoglycan Cross Linking via GlycopeptideTranspeptidase • Staphylococcus aureuscommon cause of staph infections • Peptidoglycan cell wall of polysacharides (yellow) cross-linked with tetrapeptides (red) and pentaglycine bridges (blue)

  30. Penicillin Acts as a Suicide Inhibitor

  31. Cell Wall Cross-Linking in Staphylococcus aureus What is the Cleland Representation of this reaction?

  32. Chapter 8 Problems: 1-6

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