Probing Mechanisms of Peptide Bond Formation

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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

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

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Molecular Recognition
good lv group (monitored by UV)
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Intramolecular trans-acylation reactive
thioester becomes stable amide

• Crown ether model demonstrates concepts of
• proximity of reactive functionalities
• molecular recognition

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• 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

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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

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1. Substrate (peptide) binds to form complex

1. 1st tetrahedral intermediate

1. Acyl-enzyme

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1. 2nd tetrahedral intermediate

4) H2O attacks
6) Release from enzyme
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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!