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)