chymotrypsin poster

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) His-57 acts as a general base to increase nucleophilicity of the O atom in Ser-195 (Refer to Section 4, Step 2) 2. S 1 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: Unstable tetrahedral intermediate (Refer Step 3 in the mechanism) 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 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 H 2 O 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 S 1 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) S 1 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)

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Page 1: Chymotrypsin poster

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)