1. References: Kashima A, Inoue Y, Sugio S, Madea I, Nose T and Shimohigashi Y (1998): X-ray crystal structure of a dipeptide-chymotrypsin complex in an inhibitory interaction; Khan A and James N.G.
(1998): Molecular mechanisms for the conversion of zymogens to active proteolytic enzymes; Latha B, Ramakrishnan M, Jayaraman V, Babu M (1997): Serum enzymatic changes modulated using
trypsin: chymotrypsin preparation during burn wounds in humans. Burns, 23:560-4; Polgar L (2005): The catalytic triad of serine proteases; Wenzhe Ma, Chao Tang, and Luhua Lai (2005): Specificity of
Trypsin and Chymotrypsin: Loop-Motion-Controlled Dynamic Correlation as a Determinant. Biophysical Journal. Volume 89. 1183–1193.
2. Structure
• Three chains linked by disulfide bonds (Refer Fig. 3)
• Three factors which make construct the active site of the chymotrypsin
1. Catalytic triad: There is concerted hydrogen bonding
between the residues of the triad (Polgar, 2005): the side chain of
Ser-195 is hydrogen bonded to the imidazole of the His-57, whilst the
the –NH group of this imidazole is hydrogen bonded to the carboxylate
group of Asp-102 (Refer Fig. 1)
Ø His-57 acts as a general base to increase nucleophilicity of the
O atom in Ser-195 (Refer to Section 4, Step 2)
2. S1 primary pocket: Only substrates with aromatic residues
can bind here (Refer Fig. 3) gives chymotrypsin its primary specificity
3. Oxyanion hole: Amide nitrogen from peptide backbone of
Ser-195 and Gly-193 help stabilise:
Ø Unstable tetrahedral intermediate (Refer Step 3 in the
mechanism)
Ø Transition state that proceeds formation to tetrahedral
intermediate (Wenzhe et al, 2005; Polgar, 2005)
1. Introduction
• Chymotrypsin belongs to a superfamily of serine proteases involved
in hydrolysis of peptide bonds using an active serine residue that is
part of a “catalytic triad”: Asp-102, His-57, Ser-195
• Located in the pancreas - vital for the digestion of dietary proteins
• It has a primary specificity for large, aromatic, hydrophopbic amino
acid residues (Phe, Tyr, Trp) (Wenzhe et al, 2005)
3. Regulation of Chymotrypsin
• Due to the power of proteolytic activity, premature hydrolysis must be
avoided. Chymotrypsin is initially synthesised as a zymogen called
chymotrypsinogen (Khan et al, 1998). This zymogen is activated by
proteolytic cleavage the overall structure (Refer Fig. A)
4. Catalytic Mechanism of Chymotrypsin
STEP I – ACYLATION (Polgar, 2005)
1. Substrate positioned within the active site
2. O atom on Ser-195 (Fig. 1) induces a nucleophilic
attack of Ser-195 to the carbon atom within the
carbonyl of the peptide bond
3. Unstable tetrahedral intermediate formed
Ø The transition state converts to a high energy
tetrahedral intermediate
4. An acyl-enzyme is formed as the His-57 acts as a
general base
5. An amine compound is the leaving molecule group
due to the peptide cleavage that occurs in Step 4
------------------------------------------------------------------------
STEP II – DEACYLATION (Polgar, 2005)
6. Water molecule binds onto the active site
7. His-57 now acts a general acid by drawing a proton away
from a water molecule
8. Ester group in acyl enzyme is hydrolysed
9. The O atom in H2O is a strong nucleophile
10. Repeat Step 3-4
11. A carboxylic acid compound leaves and the enzyme is
ready for the next set of catalysis
(1)
5. Future Research
• Treatment of burns by the decreasing tissue destruction
• Treatment of hand fractures to reduce redness and
inflammation (Latha et al, 1997)
Fig. 2 The overall structure of chymotrypsin
emphasising The S1 pocket is located near the
catalytic triad and Gly-193 (in green) which is
part of the oxyanion hole. PDUB 7GCH.
Fig.1 Catalytic Triad: Ser-195, His-57 and
Asp-102. The dashed lines demonstrate the
hydrogen bonding between the residues of
the catalytic triad . Due to these interactions,
the weak nucleophile of O in Ser-195 becomes
a stronger nucleophile (Kashima et al, 1998)
S1 specificity pocket
(2)
Oxyanion hole
Fig. 3 The overall
spherical structure of
chymotrypsin showing
Chains A, B and C. They
are linked by disulfide
bonds, shown in blue
(Kashima et al, 1998;
Khan et al, 1998)Chain A
Chain B
Chain C
(3)