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. 1) Substrate (peptide) binds to form complex 2)1 st tetrahedral intermediate 3)Acyl-enzyme

8. 5)2 nd tetrahedral intermediate 4) H 2 O 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  10 3 decrease in activity!