Serine proteases are used in the hydrolysis of peptide bonds and include chymotrypsin, elastase and trypsin. Proteases catalyse peptide bonds in polypeptides and proteins and according to Mathews et al (2000) serine proteases are distinct because they all have a serine residue that plays a critical role in the catalytic process. These serine proteases all have a similar three-dimensional structure with an aspartate, histidine and serine residue clustered about the active region. There is also always a pocket located close to the active region and according to Mathews et al (2000) the nature of this pocket gives each serine protease its specificity. For example, trypsin has a deep and narrow negatively charged pocket to attract positively charged long ions and chymotrypsin has a pocket that is lined with hydrophobic residues to attract hydrophobic compounds, see figure 1.
The hydrolysis of a peptide bond by chymotrypsin has six steps, see figure 2.
Firstly, the hydrophobic polypeptide substrate binds noncovalently to chymotrypsin pocket (stage 1). This bounding is very specific which places the active site of chymotrypsin very close to the carbonyl group bond to be cleaved. A proton from chymotrypsin is then transferred to the substrate and therefore leaves a negative charge on chymotrypsin (stage 2). This activated chymotrypsin is a strong nucleophile and attacks the carbonyl of the substrate, forming a tetrahedral transition state (Mathews et al, 2000). During this activated state the cleavage of the peptide bond occurs and leaves the N-terminal part of the substrate covalently bound to the enzyme (stage 3). The proton, originally on chymotrypsin, is transferred to the C-terminal fragment that was released from the cleavage of the C-N bond. In the place of the departed polypeptide a water molecule binds to the enzyme (stage 4). Mathew et al (2000) explains that the water molecule firstly displaces the C-terminal portion of the chain and then cleaves the acyl intermediate. A second tetrahedral transition state (stage 5) is formed when the water molecule transfers its proton to the histidine residue and the rest of the water molecule to the remaining substrate fragment. According to Mathew et al (2000) this process is a reversal of the formation of the initial acyl intermediate, with the water molecule playing the role of the released portion of the polypeptide chain. Finally, the second peptide fragment is released, the acyl bond is cleaved, the proton is transferred back to serine residue from the histidine residue and the enzyme returns to its original state (stage 6).
The example above shows that the hydrolysis of peptide bond cleavage by serine proteases involves stabilization of tetrahedral intermediate states. Mathew et al (2000) stated that energetic stabilization of activated states and correct positioning of reactants are important in lowering the free energy of activation.
The specificity of chymotrypsin depends entirely on which amino acid is directly on the amino-terminal side of the peptide bond to be cleaved (Berg et al, 2000). There are other proteases with more complex specificity patterns which may have additional pockets for the recognition of other residuals on the substrates.
