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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:
OO
O O
O
OO
OO
Li+ Na
+
12-crown-4 15-crown-5
binds Li+
(small cavity)
binds Na+
(medium cavity)
O
O
O
O
O
ON
R
HHH O
O
O
O
O
O
18-crown-6
binds K+
(large cavity)
Also binds ammonium ionsvia multiple H-bonds
+
• Koga chemically modified the crown ether to contain 2 thiol (SH) groups to mimic reactions on NRPS
O
O
O
O
O
O SHSH
S
O
NH
R1
peptide SH
S
O
NH
R1
peptide SHNH
HH
O
OR2
NO2
NH3
O
OR2
NO2
S
O
NH
R1
peptide SNH
HH
O
R2
OH NO2
Crown ether ring(receptor)
peptide chain
+
+
+
thioacylation
+
Molecular Recognition
good lv group (monitored by UV)
S
O
NH
R1
peptide SNH2
O
R2
-O NO2
SH
O
NH
R1
peptideNH
S
O
R2
+increase pH
Intramolecular trans-acylation: reactive thioester becomes stable amide
• Crown ether model demonstrates concepts of:
• proximity of reactive functionalities
• molecular recognition
• 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
R OH
O
NH
H
R'R NH
O
R' ++ H2O
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
O
O
N NH
His 57
H O N
O
H
O H NNH
O
O
N NH
His 57
H
O
O
N NH
His 57
NH2
O N
O
HON
HH
N
H N
O N
O
O H
Asp 102Ser 195
Gly 193
+
Asp 102Ser 195
Gly 193
Asp 102Ser 195
Gly 193
1) Substrate (peptide) binds to form complex
2) 1st tetrahedral intermediate
3) Acyl-enzyme
HO
O
N NH
His 57
OH N
O N
O
HOH
H N
O
O
N NH
His 57
O N
O
H
H
H N
O N
O
O HH
O
H
NO
O
NH
His 57
OOH
Ser 195
Gly 193
+
Ser 195
Gly 193
Asp 102Ser 195
Gly 193
5) 2nd tetrahedral intermediate
4) H2O attacks
6) Release from enzyme
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!
O P
O
O
F
Diisopropylfluorophosphate
O
O
N NH
His 57
H O
Ser
Asp 102
