Serine proteases chymotrypsin

Catalysis by enzymes that proceeds via a unique reaction mechanism typically occurs when the transition state intermediate forms a covalent bond with the enzyme (covalent catalysis). The catalytic mechanism of the serine protease chymotrypsin (Figure 7-7) illustrates how an enzyme utilizes covalent catalysis to provide a unique reaction pathway. 

Figure 7. Two examples of irreversible inactivators that are not suicide substrates a) TPCK, a classic" affinity label of the serine protease chymotrypsin, b) ZFK-CH2-mesitoate, a quiescent" affinity label of the cysteine protease cathepsin B, and c) the kinetic scheme for both forms of affinity label-inactivation.

The currently accepted mechanism for the hydrolysis of amides and esters catalyzed by the archetypal serine protease chymotrypsin involves the initial formation of a Michaelis complex followed by the acylation of Ser-195 to give an acylenzyme (Chapter 1) (equation 7.1). Much of the kinetic work with the enzyme has been directed toward detecting the acylenzyme. This work can be used to illustrate the available methods that are based on pre-steady state and steady state kinetics. The acylenzyme accumulates in the hydrolysis of activated or specific ester substrates (k2 > k3), so that the detection is relatively straightforward. Accumulation does not occur with the physiologically relevant peptides (k2 < k3), and detection is difficult. 

Within each protease family, individual members will differ in their substrate specificity. Most proteases have extended substrate binding sites and will bind to and recognize several amino acid residues of a polypeptide substrate (see Figure 2). Usually one of these will be the primary binding site. For example, in the serine proteases chymotrypsin, trypsin, and elastase, the primary substrate binding site is the Si subsite... 

Figure 7.17. Convergent Evolntion of Protease Active Sites. The relative positions of the three key residues shown are nearly identical in the active sites of the serine proteases chymotrypsin and subtilisin.

Enzyme which can hydrolyse the sericin is classified as proteolytic enzymes [63-65]. The proteolytic enzymes cleave the peptide/amide linkages and convert them into amino acid. Mainly there are three types of proteolytic enzymes such as zinc protease (e.g. carboxy peptidase A), serine protease (Chymotrypsin, Trypsin, Thrombin) and thiol protease (acts as cystine residue in the protein). The function of proteolytic enzymes in their degree of degumming depends on the pH of the bath and the optimum activity is found to be different at different pH for different enzymes. 

Figure 12.S-S. Scheme of the catalytic mechanism of serine proteases (chymotrypsin numbering).

Proteolytic enzymes (proteases) catalyze the hydrolysis of peptide bonds. The pancreatic serine proteases chymotrypsin, trypsin, and elastase have similar structures and mechanisms of action, but different substrate specificities. It is thought that they evolved from a common ancestral protease. 

The structurally modified renin inhibitor 181 is prepared by the coupling of amine (179) and the histidine derivative (180) using DEPC.70a Dipeptide is evaluated for its susceptibility to cleavage by the serine protease chymotrypsin. 

Figure 8.50 Mechanisms for two bio-cataLysts that employ nucleophilic catalysis as part of their mechanistic paths to successful bio-catalysis, (a) acetyl choline estersase. (b) serine protease chymotrypsin. All substrates are shown in red. Nucleophilic catalysis is brought about by appropriate amino acid residues that possess nucleophilic side chains for routine nucleophilic catalysis operations.

The reaction conditions can be optimized by examining the effect of different factors snch as water content, temperature, pH, surfactant concentration, reaction time, or product yield. Proteases are classified according to their catalytic mechanisms. Four mechanistic classes have been recognized by the International Union of Biochemistry and Molecular Biology serine proteases (chymotrypsin, trypsin, elastase, subtilisin), cysteine proteases (papain, cathepsins, caspases), aspartic proteases (pepsins, cathepsins, lennins), and metallo proteases. 

Fluoroalkyl P-amino alcohols are precursors of the corresponding fluoroalkyl peptidyl ketones which have been shown to be effective inhibitors of proteolytic enzymes, such as serine proteases/ (chymotrypsin/ elastases/ trypsin/ thrombin/ ) aspartyl proteasesor cysteine proteases, In some cases fluoroalkyl p-amino alcohols are themselves inhibitors of the same enzymes/ " This interest in these fluoroalkyl P-amino alcohols 1 aroused efforts for stereoselective and enantioselective synthetic methodsA number of approaches have been reported during these last years, either through the addition of trifluoromethyl equivalent anion to protected aldehyde or through the building block approach. However the main problem of all these approaches is the diastereoselectivity. 

---