Mechanism of enzyme action. Chymotrypsin

The most promising direction for enzyme modeling is to synthetically mimick the nature of the binding site and the active site in terms of the close similarity of catalytic groups, stereochemistry, interatomic distances and the mechanism of the action of the enzyme. Mimicking of the proton-transfer relay proposed for the mechanism of the action of chymotrypsin is a brilliant example of such work (D Souza and Bender, 1987 and references therein). The miniature organic model of chymotrypsin built on the basis of cyclodextrin and the mechanism of hydrolysis m-tert-butylphenyl acetate is presented in Fig. 6.9. 

How can we elucidate the role of serine 195 in chymotrypsin action A study of the enzyme s kinetics provided a second clue to chymotrypsin s catalytic mechanism and the role of serine 195. The kinetics of enzyme action are often easily monitored by having the enzyme act on a substrate analog that forms a colored product. For chymotrypsin, such a chromogenic substrate is A-acetyl-l-phenyManine />-nitrophenyl ester. This substrate is an ester rather than an amide, but many proteases will also hydrolyze esters. One of the products formed by chymotrypsin s cleavage of this substrate is p- nitrophenolate, which has a yellow color (Figure 9.3). Measurements of the absorbance of light revealed the amount of p-nitrophenolate being produced. 

The mechanism of chymotrypsin action is particularly well studied and, in many respects, typical. Numerous types of reaction mechanisms for enzyme action are known, and we shall discuss them in the contexts of the reactions catalyzed by the enzymes in question. To lay the groundwork, it is useful to discuss some general types of catalytic mechanisms and how they affect the specificity of enzymatic reactions. 

The mechanism of the action of acetylcholinesterase purified from the electric organs of Electrophorus electricus involves the attraction of the positively charged nitrogen of acetylcholine to an anionic site on the enzyme and cleavage of the substrate at an esteratic site of a nucleophilic character. The irreversible inhibition by the alkyl phosphates, tetraethyl pyrophosphate (TEPP) and diisopropyl-fluorophosphate (DFP) may be due to phosphorylation of the nucleophilic esteratic site. The phosphorylation by DFP of the phenolic hydroxyl group of free tyrosine has been demonstrated by Ashbolt and Rydon. Chymotrypsin and citrus fruit acetylesterase are also inhibited by DFP and TEPP. ... 

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. 

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... 

In subsequent years, much evidence has been adduced to support this mechanism. Alkaline phosphatase and, by analogy, other serine enzymes, are directly phosphorylated on serine serine phosphate is not an artifact (Kennedy and Koshland, 1957). In the presence of nitrophenyl acetate, chymotrypsin is acetylated on serine, and the resulting acetylchymotrypsin has been isolated (Balls and Aldrich, 1955 Balls and Wood, 1956). Similarly, the action of p-nitrophenyl pivalate gave rise to pivaloyl chymotrypsin, which could be crystallized (Balls et al., 1957). Neurath and workers showed that acetylchymotrypsin is hydrolyzed at pH 5.5, but that it is reversibly denatured by 8 M urea the denatured derivative is inert to hydrolysis and even to hydroxylamine, whereas the renatured protein, obtained by... 

The mechanism of action of anticholinesterases is to form a stable covalent complex with the Achase enzyme. Achase is one of several enzymes known as serine esterases. Other examples include the intestinal enzymes trypsin and chymotrypsin as well as the blood clotting agent thrombin. During the course of the catalysis the alcohol -OH of a serine side chain in the active site of the enzyme forms an ester complex, called the acyl-enzyme, with the substrate. So, acetylcholine will go through similar chemical reactions with Achase. 

The probable mechanism of action of chymotrypsin. The six panels show the initial enzyme-substrate complex (a), the first tetrahedral (oxyanion) intermediate (b), the acyl-enzyme (ester) intermediate with the amine product departing (c), the same acyl-enzyme intermediate with water entering (d), the second tetrahedral (oxyanion)... 

As an example, let s consider the mechanism of action of the enzyme chymotrypsin. This enzyme is known as a serine protease because it catalyzes the hydrolysis of... 

Proteolytic enzymes, such as the serine proteases, are among the best characterized of all enzymes.They are important in digestive processes because they break down proteins. They each catalyze the same type of reaction, that is. the breaking of peptide bonds by hydrolysis. The crystal structures of several serine proteases have been determined, and the mechanism of hydrolysis is similar for each. The specificity of each enzyme is, however, different and is dictated by the nature of the side chains flanking the scissile peptide bond (the bond that is broken in catalytic mechanism. Chymotrypsin is one of the best characterized of these serine proteases. The preferred substrates of chymotrypsin have bulky aromatic side chains. The crystal structure determination of the active site of chymotrypsin, illustrated in Figure 18.12, has provided much of the information used to elucidate a plausible mechanism of action of the enzyme. In the first step of any catalyzed reaction, the enzyme and substrate form a complex, ES, the Michaelis complex. The hydrolysis of the peptide bond by chymotrypsin involves three amino acid residues,... 

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 determination of the three-dimensional structure of chymotrypsin by David Blow in 1967 was a source of further insight into its mechanism of action. Overall, chymotrypsin is roughly spherical and comprises three polypeptide chains, linked by disulfide bonds. It is synthesized as a single polypeptide, termed chymotrypsinogen, which is activated by the proteolytic cleavage of the polypeptide to yield the three chains. The active site of chymotrypsin, marked by serine 195, lies in a cleft on the surface of the enzyme (Figure 9.6). 

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