Chymotrypsin enzyme mechanism

We present here the mechanisms for four enzymes chymotrypsin, hexoldnase, enolase, and lysozyme. These examples are not intended to cover all possible classes of enzyme chemistry. They are chosen in part because they are among the best understood enzymes, and in part because they clearly illustrate some general principles outlined in this chapter. The discussion concentrates on selected principles, along with some key experiments that have helped to bring these principles into focus. We use the chymotrypsin example to review some of the conventions used to depict enzyme mechanisms. Much mechanistic detail and experimental evidence is necessarily omitted no one book could completely document the rich experimental history of these enzymes. Also absent from these discussions is the special contribution of coenzymes to the catalytic activity of many enzymes. The function of coenzymes is chemically varied, and we describe each as it is encountered in Part II. 

Redesigned and Expanded Treatment of Enzyme Mechanisms NEW Mechanism Figures designed to lead students through these reactions step by step. The first reaction mechanism treated in the book, chymotrypsin, presents a refresher on how to follow and understand reaction mechanism diagrams. Twelve new mechanisms have been added, including lysozyme. 

J. Fastrez and A.R. Fersht. 1973. Demonstration of the acyl-enzyme mechanism for the hydrolysis of peptides and anilides by chymotrypsin TJiocAe/wAfry 12 2025-2034. (PubMed)... 

If the a-chymotrypsin-catalysed hydrolysis of 4-nitrophenyl acetate [10] is monitored at 400 nm (to detect 4-nitrophenolate ion product) using relatively high concentrations of enzyme, the absorbance time trace is characterised by an initial burst (Fig. 5a). Obviously the initial burst cannot be instantaneous and if one uses a rapid-mixing stopped-flow spectrophotometer to study this reaction, the absorbance time trace appears as in Fig. 5b. Such observations have been reported for a number of enzymes (e.g. a-chymotrypsin [11], elastase [12], carboxypeptidase Y [13]) and interpreted in terms of an acyl-enzyme mechanism (Eqn. 7) in which the physical Michaelis complex, ES, reacts to give a covalent complex, ES (the acyl-enzyme) and one of the products (monitored here at 400 nm). This acyl-enzyme then breaks down to regenerate free enzyme and produce the other products. The dissociation constant of ES is k2 is the rate coefficient of acylation of the enzyme and A 3 is the deacylation rate coefficient. Detailed kinetic analysis of this system [11] has shown... 

This Highlight is part of an extraordinary story (also a cautionary tale) in the area of biocatalysis. The point of particular interest was the incredible catalytic activity claimed for so-called pepzymes - small synthetic peptides modelled to mimic the active site structures of trypsin and chymotrypsin. One was claimed to hydrolyse a simple peptide (a trypsin substrate) with efficiency comparable to that of the native enzyme. This extraordinary result provoked at least as much scepticism as excitement, and in the following months several groups tried to reproduce the results. They failed, comprehensively. [1,2] Some reasons why this failure came as no surprise were subsequently summarised by Matthews, Craik and Neurath, [3] and by Corey and Corey [4]. The background has been discussed in an Angewandte Review on Enzyme Mechanisms, Models and Mimics. [5]... 

In the case of enzymes reacting at measurable rates with a wide variety of substrates, structure-reactivity correlations are useful to establish mechanistic similarities with model reactions involving proton transfers [11]. As with most other methods applied to enzyme mechanisms, use of this criterion alone can be misleading. For a-chymotrypsin, for example, a limited series of substrates can be found which shows reactivities not inconsistent with the active-site imidazole acting as a nucleophile [12], whereas overwhelming evidence from all other methods shows that the imidazole acts as a general base [13,14]. 

The duality of hydrolytic enzyme mechanisms, chymotrypsin and carboxypeptidase-, and that of decarboxylation by metal ion and... 

An example of a pseudoirreversible inhibitor has been demonstrated for chymotrypsin (36). This enzyme is a serine protease, and its mechanism of catalysis may be outlined as follows, where or R2 preferentially is a hydrophobic amino acid residue. 

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. 

The catalytic mechanism of the subtilisins is the same as that of the digestive enzymes trypsin and chymotrypsin as well as that of enzymes in the blood clotting cascade, reproduction and other mammalian enzymes. The enzymes are known as serine proteases due to the serine residue which is crucial for catalysis (Kraut, 1977 and Polgar, 1987)... 

Kirsh et al. 42) prepared apolar derivatives of poly(4-vinylpyridine) by benzylation. With nitrophenyl acetate as the substrate the benzylated catalyst is 100 times more effective than 4-ethylpyridine. A double-displacement mechanism was observed. The rate constants for deacylation of the acylpoly(vinylpyridine) derivatives were about 4 x 10" /sec. The comparable value for a-chymotrypsin is 8 x 10 /sec. The factor of 20 seems small, but it should be kept in mind that deacetylation of a-chymotrypsin is very slow compared with the deacylation reactions involving the natural substrates of the enzyme. 

However, not included in the above mechanisms are other amino acid side-chains at the active site, whose special role will be to help bind the reagents in the required conformation for the reaction to occur. Examples of such interactions are found with acetylcholinesterase and chymotrypsin, representatives of a group of hydrolytic enzymes termed serine hydrolases, in that a specific serine amino acid residue is crucial for the mechanism of action. 

Acetylcholine is a relatively small molecule that is responsible for nerve-impulse transmission in animals. As soon as it has interacted with its receptor and triggered the nerve response, it must be degraded and released before any further interaction at the receptor is possible. Degradation is achieved by hydrolysis to acetate and choline by the action of the enzyme acetylcholinesterase, which is located in the synaptic cleft. Acetylcholinesterase is a serine esterase that has a mechanism similar to that of chymotrypsin (see Box 13.5). 

For example, chymotrypsin cleaves peptides on the C-terminal side of aromatic amino acid residues phenylalanine, tyrosine, and tryptophan, and to a lesser extent some other residues with bulky side-chains, e.g. Leu, Met, Asn, Gin. On the other hand, trypsin cleaves peptides on the C-terminal side of the basic residues arginine and lysine. Elastase usually catalyses hydrolysis of peptide bonds on the C-terminal side of neutral aliphatic amino acids, especially glycine or alanine. These three pancreatic enzymes are about 40% identical in their amino acid sequences, and their catalytic mechanisms are nearly identical. 

The mechanism of action of chymotrypsin can be rationalized as follows (Figure 13.5). The enzyme-substrate complex forms, with the substrate being positioned correctly through hydrogen bonding and interaction with the pocket as described above. The nucleophilicity of a serine residue is only modest, but here it is improved by... 

Figure 3-2. Reaction mechanism of chymotrypsin as an example of covalent catalysis. Step I involves attack of the enzyme s active site serine on the peptide bond to be cleaved. In step II, a covalent complex is formed between the enzyme and a portion of the substrate (peptide 2) with release of the rest of the substrate (peptide I). Step III involves hydrolysis of the enzyme-substrate complex, which releases peptide 2 and completes the reaction.

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