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Probing Mechanisms of Peptide Bond Formation & Catalysis Using Models

Probing Mechanisms of Peptide Bond Formation & Catalysis Using Models. Model of Koga Uses molecular recognition by a crown ether to bind a model of the substrate Crown ethers:. Koga chemically modified the crown ether to contain 2 thiol (SH) groups to mimic reactions on NRPS.

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Probing Mechanisms of Peptide Bond Formation & Catalysis Using Models

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  1. Probing Mechanisms of Peptide Bond Formation & Catalysis Using Models • Model of Koga • Uses molecular recognition by a crown ether to bind a model of the substrate • Crown ethers:

  2. Koga chemically modified the crown ether to contain 2 thiol (SH) groups to mimic reactions on NRPS

  3. Molecular Recognition good lv group (monitored by UV)

  4. Intramolecular trans-acylation: reactive thioester becomes stable amide • Crown ether model demonstrates concepts of: • proximity of reactive functionalities • molecular recognition

  5. We have examined peptide bond formation on the ribosome • Catalytic mechanism: • The ribosome “workbench” • rRNA • Substrate-assisted catalysis • What about breaking the peptide bond (i.e. proteolysis)? • Catalyzed by proteases and/or peptidases

  6. Chymotrypsin • Part of the serine proteases (trypsin, elastase, etc) • These enzymes often work in concert • Serine residue in active site acts as nucleophile • Chymotrypsin cuts peptide bonds at aromatic residues

  7. Substrate (peptide) binds to form complex • 1st tetrahedral intermediate • Acyl-enzyme

  8. 2nd tetrahedral intermediate 4) H2O attacks 6) Release from enzyme

  9. Test of Mechanism? • How do we know Ser is the original nucleophile? • Use irreversible inhibitor to react with Ser • Ser 195 is more nucleophilic than other serine residues • Nearby His 57 & Asp 102 are important for catalytic activity. How? • Methylate His nitrogen  103 decrease in activity!

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