Chymotrypsin catalytic mechanism

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)... 

The group is typically divided into two families the subtilisin-like and chymotrypsin-like groups (7). The chymoti sin-like group are represented by members in all organisms whereas the subtilisin-like members are found in only prokaryotes. Although both families have dissimilar primary and tertiaiy structures they share a common catalytic mechanism (8,9),... 

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. 

Induced fit is only one aspect of the catalytic mechanism of hexokinase—like chymotrypsin, hexokinase uses several catalytic strategies. For example, the active-... 

The most-studied enzyme in this context is chymotrypsin. Besides being well characterized in both its structure and its catalytic mechanism, it has the advantage of a very broad specificity. Substrates may be chosen to obey the simple Michaelis-Menten mechanism, to accumulate intermediates, to show nonproductive binding, and to exhibit Briggs-Haldane kinetics with a change of rate-determining step with pH. 

The sequence of a protein of interest can be compared with all other known sequences to ascertain whether significant similarities exist. Does this protein belong to one of the establishedfamiliesl A search for kinship between a newly sequenced protein and the thousands of previously sequenced ones takes only a few seconds on a personal computer (Section 7.2). If the newly isolated protein is a member of one of the established classes of protein, we can begin to infer information about the protein s function. For instance, chymotrypsin and trypsin are members of the serine protease family, a clan of proteolytic enzymes that have a common catalytic mechanism based on a reactive serine residue (Section 9.1.4). If the sequence of the newly isolated protein shows sequence similarity with trypsin or chymotrypsin, the result suggests that it may be a serine protease. 

What is the nucleophile that chymotrypsin employs to attack the substrate carbonyl group A clue came from the fact that chymotrypsin contains an extraordinarily reactive serine residue. Treatment with organofluorophosphates such as diisopropylphosphofluoridate (DIPF) (Section 8.5.2) was found to inactivate the enzyme irreversibly (Figure 9.2). Despite the fact that the enzyme possesses 28 serine residues, only one, serine 195, was modified, resulting in a total loss of enzyme activity. This chemical modification reaction suggested that this unusually reactive serine residue plays a central role in the catalytic mechanism of chymotrypsin. 

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. 

FiGDRE 2.S3 Catalytic mechanism for hydrolysis of a peptide bond by chymotrypsin. fa) Four amino adds in the polypeptide chain of chymotrypsin have been shown ro participate in the chemLiitry of catalysis His S7, Asp 102, Gly 193, and Ser 195. (b Binding of the substrate a dietary polypeptide, to the active site of chymotrypsin. (c> The carbonyl group of the target peptide bond of the substrate becomes more polarized. (dl A charge relay... 

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,... 

Chymotrypsin and subtilisin also differ in their amino acid sequences, number of disulfide bridges (chymotrypsin has five, whereas subtilisin has none), and overall three-dimensional structures. The striking difference in structure and common catalytic mechanism are taken as evidence of an independent but convergent evolutionary process. 

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

In further studies of a-chymotrypsin, Rajender, Han, and Lumry have been able to estimate the standard enthalpies and entropies of formation of the intermediates in the catalytic mechanism given in (1) when applied to V-acetyl-L-tryptophanethylester, which is a good substrate, that is, a substrate rapidly hydrolyzed by this enzyme. The analysis, in addition to the assumption that (1) is valid, requires cancellation of pH dependencies of the individual rate constants. The latter is not quite complete so that the computed quantities are unreliable at pH values above 9. Nevertheless, they are adequate to show that the same compensation pattern found with inhibitors occurs in normal catalysis. These results are shown in Fig. 3, in which segments of the compensation line for indole binding obtained by Yapel and Lumry are given for reference. Rajender et al. also found linear compensation... 

The catalytic strategies employed by chymotrypsin to increase the reaction rate are common to many enzymes. One of these catalytic strategies, proximity and orientation, is an intrinsic feature of substrate binding and part of the catalytic mechanism of all enzymes. All enzymes also stabilize the transition state by electrostatic interactions, but not all enzymes form covalent intermediates. 

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. 

Acyl enzyme, an intermediate in the catalytic mechanism of serine proteases, such as trypsin and chymotrypsin. After the serine protease has bound a peptide substrate to form the Michaelis complex, Ser (in the case of chymotrypsin) nucleophilically attacks the peptide bond in the rate-determining step, forming a transition-state complex, known as a tetrahedral intermediate. The latter decomposes to the acyl enzyme, an extremely unstable intermediate, that bears the acyl moiety at the hydroxy group of Ser . The acyl enzyme intermediate is deacylated by water during proteolysis, or the attacking nucleophile is an amino component in case of kineticaUy controlled enzymatic peptide synthesis. 

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