Chymotrypsin peptide hydrolysis

S. A. Bizzozero, H. Dutler, Stereochemical Aspects of Peptide Hydrolysis Catalyzed by Serine Proteases of the Chymotrypsin Type , Bioorg. Chem. 1981, 10, 46 - 62 ... 

Fersht, A. R., D. M. Blow, and J. Fastrez, Leaving group specificity in chymotrypsin-catalyzed hydrolysis of peptides A stereochemical interpretation. Biochem. 12 2035, 1973. 

The stereochemical aspects of peptide hydrolysis catalyzed by chymotrypsin and related serine proteases has been recently analyzed with respect to requirements for stereoelectronic control of bond cleavage and this analysis has led to a much more complete understanding of the reaction mechanism (9-14). 

The specificity of chymotrypsin for hydrolysis of peptide bonds formed by the carbo,xyl groups of tyrosine, phenylalanine, and tryptophan has been recognized for some time (Green and Neurath, 1954 Desnuelle, 1960). Action on synthetic substrates of leucine (Goldenberg et al., 1951) and methionine (Kaufman and Neurath, 1949) also has been noted although at much slower rates than observed with the aromatic amino acid derivatives. When protein substrates or synthetic ester substrates are examined, it is evident that a variety of bonds can be hydrolyzed by chymotrypsin. Inagami and Sturtevant (1960) observed that chymotryptic hydrolysis of a-benzoyl-L-arginine ethyl ester, a typical trypsin substrate, occurred at a maximum rate which was 20% of that observed with trypsin. Several ester substrates, such as p-nitrophenylacetate (Hartley and Kilby, 1954), are also hydrolyzed. 

Specificity of Chymotrypsin for Hydrolysis of Peptide Bonds in Proteins and Polypeptides ... 

Figure 9.8. Peptide Hydrolysis by Chymotrypsin. The mechanism of peptide hydrolysis illustrates the principles of covalent and acid-base catalysis. The dashed green lines indicate favorable interactions between the negatively charged aspartate residue and the positively charged histidine residue, which make the histidine residue a more powerful base.

Initial attempts to achieve an enzyme-catalyzed deprotection of the carboxy group of peptides centred around the use of the endopeptidases chymotrypsin, trypsin,and thermolysin.P l Thermolysin is a protease obtained from Bacillus thermoproteolyticus that hydrolyzes peptide bonds on the annino side of the hydrophobic amino acid residues (e.g., leucine, isoleucine, valine, phenylalanine). It cleaved the supporting tripeptide ester H-Leu-Gly-Gly-OEt from a protected undecapeptide (pH 7, rt). The octapeptide, thus obtained, is composed exclusively of hydrophilic annino acids. Due to the broad substrate specificity of thermolysin and the resulting possibility of unspecific peptide hydrolysis, this method is of limited application. 

Zamai et al. from the same company have also published data on sequence-directed peptide inhibitors [80]. They used the model of big ET-1 which they proposed on the basis of computerized structure prediction, molecular mechanics minimization and molecular dynamics [81]. The effect of the sequence-directed inhibitors on the in vitro chymotrypsin-catalyzed hydrolysis of big ET-1 was investigated as a strategy for inhibiting formation of endothelin. 

Proteins are hydrolyzed very slowly with storage in water at neutral pH. However, addition of proteases can increase the rate of hydrolysis about 10 billion times over the spontaneous rate. The chymotrypsin mechanism depicted in Figure 2.53 is shared by trypsin and elastase. These three proteases are members of a family called the serine proteases (named after Ser 195), Carboxypeptidase Aand pepsin catalyze peptide hydrolysis by different mechanisms and are not part of this family. 

As with peptide hydrolysis, several enzyme systems exist that catalyze carboxylic and phosphoric ester hydrolysis without the need for a metal ion. They generally involve a serine residue as the nucleophile in turn, serine may be activated by hydrogen-bond formation—or even proton abstraction—by other acid-base groups in the active site. The reaction proceeds to form an acyl- or phosphory 1-enzyme intermediate, which is then hydrolyzed with readdition of a proton to the serine oxygen. Mechanisms of this type have been proposed for chymotrypsin. In glucose-6-phosphatase the nucleophile has been proposed to be a histidine residue. ... 

The mechanism for bovine chymotrypsin-catalyzed hydrolysis of a peptide bond is shown in Figure 24.7. The other serine proteases follow the same mechanism. The reaction proceeds as follows ... 

Proposed mechanism for the chymotrypsin-catalyzed hydrolysis of a peptide bond. 

Amino-acid Protection. - Carboxyamidomethyl (CAM) esters have been shown to be useful protecting groups in a-chymotrypsin and papain-catalysed peptide hydrolysis and synthesis.5 10 1,3-Dioxans (582) can be obtained from Na-protected serine derivatives by acid-catalysed transacetalation and are sufficiently robust to survive both amino deprotection and peptide coupling reactions. 11 (L)-Histidine benzyl ester can be prepared as the ditosylate salt... 

Chymotrypsin catalyzes the hydrolysis of peptide bonds adjacent to aromatic amino acid residues in the protein being hydrolyzed other residues are attacked at a lower frequency. In addition, chymotrypsin catalyzes the hydrolysis of esters in model studies in the laboratory. The use of model systems is common in biochemistry because a model provides the essential features of a reaction in a simple form that is easier to work with than the one found in nature. The amide (peptide) bond and the ester bond are similar enough that the enzyme can accept both types of compounds as substrates. Model systems based on the hydrolysis of esters are frequendy used to study the peptide hydrolysis reaction. 

Bizzozero SA, Duller H (1981) Stereochemical aspects of peptide hydrolysis catalyzed by serine proteases of the chymotrypsin type. Bioorgan Chem 10 46-62... 

A-a-decyL-L- phenylalanine-(2-aminopyridine)amide can be regarded as a surfactant with a headgroup imitating the transition state of the a-chymotrypsin catalyzed hydrolysis of peptides. Using this TSA as a template, catalytically active silica particles were obtained. To test the reactivity, the hydrolysis of an arginine derivative was investigated (Fig. 18b). Compared to the control material prepared without template, a rate enhancement of a factor of 5 was observed. Furthermore, the catalytic silicas were shown to be enantioselective. Remarkably, the preferential... 

Chymotrypsin is a digestive enzyme and belongs to the family of serine proteases and catalyzes the hydrolysis of proteins and peptides. A part of the substrate is bound covalently to the enzyme. Chymotrypsin hydrolyzes esters as well even if this is physiologically not important. But this fact was of interest for mechanistic studies of chymotrypsin-catalyzed hydrolysis (Figure 23b). The rate-determining step is the hydrolysis of the enzyme-acetyl complex by... 

S. A. Bizzozero and B. O. Zweifel (1975), The importance of the conformation of the tetrahedral intermediate for the a-chymotrypsin-catalyzed hydrolysis of peptide substrates. FEBS Lett. 59, 105-108. 

Chymotrypsin-catalyzed hydrolysis of peptides proceeds in four steps. In the first step, the peptide linkage is attacked by a hydroxyl group on serine to form a tetrahedral intermediate (Figure 23.39). Loss of the amine from the tetrahedral intermediate gives an ester involving the... 

FIGURE 23.40 Step 2 of chymotrypsin-catalyzed hydrolysis of peptides. 

If the enzyme-catalyzed hydrolysis of peptide bond involves a simple reversible reaction as shown by Equation 2.5 then, indeed, the enzyme must catalyze the rate of formation of peptide bond from amino acids (i.e., lq,-step), provided the amino acids do not react irreversibly with the enzyme. Incidentally, if the function of serine proteases is to catalyze both the rate of hydrolytic cleavage and the rate of formation of protein peptide bond, then, probably, these enzymes cannot digest the proteins that we eat and, consequently, the results would have been disastrous for all protein-eating creatures — which certainly Nature will never allow. Although the mechanisms of most of the enzyme-catalyzed reactions are unknown, even at a very rudimentary level, the mechanism of a-chymotrypsin-catalyzed hydrolysis of peptide bond has been relatively well understood. The reaction has been almost ascertained to involve acylation and deacylation of enzyme as shown by Equation 2.6. Widely accepted mechanisms for acylation and deacylation steps are shown in Scheme 2.6 and Scheme 2.7. ... 

Chymotrypsin (Section 27 10) A digestive enzyme that cat alyzes the hydrolysis of proteins Chymotrypsin selectively catalyzes the cleavage of the peptide bond between the car boxyl group of phenylalanine tyrosine or tryptophan and some other ammo acid... 

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