Enzymes 2-chymotrypsin

Figure 11.7 Schematic diagram of the structure of chymotrypsin, which is folded into two antiparallel p domains. The six p strands of each domain are red, the side chains of the catalytic triad are dark blue, and the disulfide bridges that join the three polypeptide chains are marked in violet. Chain A (green, residues 1-13) is linked to chain B (blue, residues 16-146) by a disulfide bridge between Cys 1 and Cys 122. Chain B is in turn linked to chain C (yellow, residues 149-245) by a disulfide bridge between Cys 136 and Cys 201. Dotted lines indicate residues 14-15 and 147-148 in the inactive precursor, chmotrypsinogen. These residues are excised during the conversion of chymotrypsinogen to the active enzyme chymotrypsin.

This is nicely illustrated by members of the chymotrypsin superfamily the enzymes chymotrypsin, trypsin, and elastase have very similar three-dimensional structures but different specificity. They preferentially cleave adjacent to bulky aromatic side chains, positively charged side chains, and small uncharged side chains, respectively. Three residues, numbers 189, 216, and 226, are responsible for these preferences (Figure 11.11). Residues 216... 

Many enzymes have absolute specificity for a substrate and will not attack the molecules with common structural features. The enzyme aspartase, found in many plants and bacteria, is such an enzyme [57], It catalyzes the formation of L-aspartate by reversible addition of ammonia to the double bond of fumaric acid. Aspartase, however, does not take part in the addition of ammonia to any other unsaturated acid requiring specific optical and geometrical characteristics. At the other end of the spectrum are enzymes which do not have specificity for a given substrate and act on many molecules with similar structural characteristics. A good example is the enzyme chymotrypsin, which catalyzes hydrolysis of many different peptides or polypeptides as well as amides and esters. 

Total PA activity Total PA activity Collagenase 1 Collagenase 1 CB-Iike enzyme Trypsin-like enzyme Chymotrypsin-like enzyme 92-kDa gelatinase Heparanase... 

The repetitive cycle to identify a sequence of N-terminal amino acids has been automated. In practice, it is limited to about 20-30 amino acids, since impurities build up and the reaction mixture becomes too complex to yield unequivocal results. The usual approach is to break the polypeptide chain into smaller fragments by partial hydrolysis, preferably at positions relating to specific amino acid residues in the peptide chain. There are ways of doing this chemically, and the enzymes chymotrypsin... 

The digestive enzyme chymotrypsin has a serine in its active site that acts as a general base or proton acceptor during hydrolysis of peptide bonds in protein substrates (Figure 3-2). 

The relationship between the structure of the oligopeptide sequences and the rate of enzymatically catalyzed release of a drug or drug model was studied in detail. Over 50 different oligopeptide sequences were introduced into HPMA copolymers and their degradability by different enzymes studied. First, model enzymes, chymotrypsin [235, 238,239, 243,244], trypsin [245], and papain... 

Values of a catalytic constant, 27 k computed from (6) are listed in Table V. It is obvious that the imidazole-substituted dodecylpoly-enimines are more than 100 times as effective as the simple imidazole28,29 molecule itself. The catalytic constant for the imidazole-dodecyl polymer in fact approaches that of the enzyme chymotrypsin. 

Figure 3. Possible mechanism for (a) formation and (b) breakdown of acyl-enzyme (chymotrypsin) intermediate (3)...

Hydrolysis of peptides or proteins with acid yields a mixture of free a-amino acids. When completely hydrolyzed, each type of protein yields a characteristic proportion or mixture of the different amino acids. The 20 common amino acids almost never occur in equal amounts in a protein. Some amino acids may occur only once or not at all in a given type of protein others may occur in large numbers. Table 3-3 shows the composition of the amino acid mixtures obtained on complete hydrolysis of bovine cytochrome c and chymotrypsinogen, the inactive precursor of the digestive enzyme chymotrypsin. These two proteins, with very different functions, also differ significantly in the relative numbers of each kind of amino acid they contain. 

The enzyme chymotrypsin, with bound substrate in red (PDB ID 7GCH). Some key active-site amino acid residues appear as a red splotch on the enzyme surface. 

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. 

An excellent example of this effect is seen in the kinetics associated with a series of related substrates for the enzyme chymotrypsin (Fig. 1). Chymotrypsin... 

Figure 9-3 pH dependence of the rate of cleavage of an amide bond by the enzyme chymotrypsin, which helps digest proteins in your intestine. [M. L Bender. G. C. Clement, F. J. Kezdy, and H. A Heck. The Correlation of the pH (pD) Dependence and the Stepwise Mechanism of <,Chymotrypsin-Catalyzed-Reactions."J. Am. Chem. Soc. 1964,86.3680. ... 

In the acylation step a nucleophilic group on one of the amino-acid side chains at the active site behaves as the nucleophile. As we have seen in Section 25-9B, the nucleophile of carboxypeptidase is the free carboxyl group of glutamic acid 270. In several other enzymes (chymotrypsin, subtilisin, trypsin, elastase, thrombin, acetylcholinesterase), it is the hydroxyl group of a serine residue ... 

Chapter 8, How Enzymes Work, starts with a description of the basic chemical mechanisms that are exploited by enzymes. The latter half of this chapter presents a detailed description of how three enzymes—chymotrypsin, RNase, and triose phosphate isomerase—exploit these basic mechanisms of enzyme catalysis. 

One of the most investigated type of reaction in the field of catalytic imprinted polymers, as indicated by the large number of publications available, is certainly ester hydrolysis. In particular, a great deal of work has been carried out on systems inspired by hydrolytic enzymes since 1987. In 2000, Shea et al. [37] reported the preparation of enantioselective imprinted polymers for the hydrolysis of N-tert-butoxycarbonyl phenylalanine-p-nitrophenyl ester (55), using a system already developed by the same group in 1994 [19]. The system was inspired by the natural hydrolytic enzyme chymotrypsin and polymerisable imidazole units (27) were used as functional monomers coupled via ester linkages to a chiral phosphonate (56), analogue of (d)- or (L)-phenyl-alanine. After template removal, the imprinted polymers showed selectivity towards the hydrolysis of the enantiomer with which they were imprinted. The ratio of the rate constants, k /k, was 1.9 for the polymer imprinted with the D-enantiomer and kjku was 1.2 for that imprinted with the L-enantiomer. Moreover, the imprinted polymer showed a 2.5-fold increase in the rate of the reaction when compared with the control polymer, imprinted with a... 

Organophosphorus compounds bearing a fluorescent group were specifically introduced into the active sites of the serine-enzymes -Chymotrypsin, Trypsin and Butyrylcholin-esterase using the agents 2, 3i and 4. This was shown using electrophoresis. 

The compounds 2, 3 and 4 are effective inhibitors of serine enzymes ( -Chymotrypsin, Trypsin, Butyrylcholin-esterase and Acetylcholin-esterase (only 4). 

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... 

Drags that structurally resemble nutrients such as polypeptides, nucleotides, or fatty acids may be especially susceptible to enzymatic degradation. For example, the proteolytic enzymes chymotrypsin and trypsin can degrade insulin and other peptide drags. In the case of insulin, proteolysis was shown to be reduced by the coadmmistration of carbopol polymers at 1% and 4% (w/v%), which presumably shifted the intestinal pH away from the optimal pH for proteolytic degradation. 

In a chemical enzyme-modification experiment conducted by E. F. Jansen and colleagues in 1949, chymotrypsin was incubated with 32P-Iabeled DFP and then hydrolyzed with a strong acid. Separation of the constituent amino acids revealed 1 mol of labeled Ophosphorylserine per 25,000 g of chymotrypsin. Since DFP is a potent inhibitor of the enzyme chymotrypsin, what might we infer about the amino acid side-chain composition of the active site ... 

During last decades the domains C-2 symmetry (the dyad rotation symmetry) of low-B palindrome was established in many enzymes (chymotrypsin, trypsin, aspartyl proteinases, HIV-1 protease, carboxypeptidase A, phospholipase A-2 ribonuclease, etc.) (Lumry, 2002 and references therein). It is proposed that the pair domain closure causes constrain of pretransition state complex that activates cleavage or formation of chemical bonds. Thus control of strong bonds by the cooperation of many matrix or knots bonds takes place. As an example, in the active site of carboxypeptidase A the zinc ion is attached to one of the catalytic domains by histidine 69 and glutamine 72 and connected by hystidine 196 to the second domain. Similar structures were found in the chymotrypsin and pepsin active sites where protons are driven under compression of the domains closure. 

I Cyclodextrins are excellent enzyme models Catalysis and induced fit. Due to their cavities, which are able to accommodate guest (substrate) molecules, and due to the many hydroxyl groups lining this cavity, cyclodextrins can act catalytically in a variety of chemical reactions and they therefore serve as good model enzymes. Thus, benzoic acid esters are hydrolyzed in I aqueous solution by factors up to 100 times faster if cyclodextrins are added. The reaction in- j volves an acylated cyclodextrin as intermediate which is hydrolyzed in a second step of the j reaction, a mechanism reminiscent of the enzyme chymotrypsin. The catalytic efficiency can. be further enhanced if the cyclodextrins are suitably modified chemically so that a whole range of artificial enzymes have been synthesized [551-555, 556, 563, 564]. 

Figure 8.21. Affinity Labeling. (A) Tosy 1-1-phenylalanine chloromethyl ketone (TPCK) is a reactive analog of the normal substrate for the enzyme chymotrypsin. (B) TPCK binds at the active site of chymotrypsin and modifies an essential histidine residue.

Covalent catalysis. In covalent catalysis, the active site contains a reactive group, usually a powerful nucleophile that becomes temporarily covalently modified in the course of catalysis. The proteolytic enzyme chymotrypsin provides an excellent example of this mechanism (Section 9.1.2). 

A number of proteolytic enzymes participate in the breakdown of proteins in the digestive systems of mammals and other organisms. One such enzyme, chymotrypsin, cleaves peptide bonds selectively on the carboxylterminal side of the large hydrophobic amino acids such as tryptophan, tyrosine, phenylalanine, and methionine (Figure 91). Chymotry psin is a good example of the use of covalent modification as a catalytic strategy. The enzyme employs a powerful nucleophile to attack the unreactive carbonyl group of the substrate. This nucleophile becomes covalently attached to the substrate briefly in the course of catalysis. 

Figure 7-10 shows ithe schematic of the enzyme chymotrypsin. In many cases the enzyme s active catalytic sites are found where the various loops interact. For chymotrypsin the catalytic sites are noted by the numbers 57,102, and 195 in Figure 7-10. A number of structures of enzymes or pertinent information can be found on the following WWW sites ...

Most of the earlier studies on the immobilization of enzymes were directed towards the attachment of the enzymes to water-insoluble polymeric supports such as cellulose dextran derivatives, polyacrylamide and porous glass Diffusion problems and steric hindrance are two main factors affecting the application of such supports. The introduction of soluble polymers for immobilization purposes overcomes these difficulties to a greater extent. These soluble enzyme derivatives were synthesized in order to increase the effective molecular size of parent en mes this would rmit the use of ultrafiltration without any los of the enzyme. O NeiD etal. immobilized the enzyme chymotrypsin on soluble dextran for... 

The enzyme chymotrypsin has been used for enzymatic zonulolysis during operations for cataract (1). Its possible role in precipitating postoperative intraocular hypertension has been studied, but the use of enzymatic... 

Figure 37.2. Catalysis by the enzyme chymotrypsin of the cleavage of one peptide bond in a protein a proposed mechanism. Histidine and pro-tonated histidine act as general base and acid in two successive nucleophilic substitution reactions (a) cleavage of protein with formation of acyl enzyme and liberation of one protein fragment (6) hydrolysis of acyl enzyme with regeneration of the enzyme and liberation of the other protein fragment.

We have noted previously that the catalytic effect of the hydrolytic enzyme chymotrypsin depends critically on the interaction of a hydroxyl and an imidazole group placed in juxtaposition in the enzyme molecule. It was, therefore, particularly tempting to see if an analogous interaction could enhance the catalytic efficiency of PVI, by using copolymers of 4(5)-vinylimidazole with p-vinylphenol (VI/VP) or with vinyl alcohol (VI/VA)... 

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