Chymotrypsin specificity

Chymotrypsin specifically cleaves peptide bonds whose C-terminal amino acid is adjacent to non-polar aromatic amino acid residues or the bulky, hydrophobic methionine. Because these residues are often buried in the interior of proteins, including chymotrypsin, the self-hydrolysis of native, folded chymotrypsin is very inefficient. In fact, during digestion, chymotrypsin acts most effectively on partially degraded and denatured (unfolded) proteins. 

Leupeptin, trypsin inhibitor of actinomycetes, inhibited the esterase activity of Cls for arginine as well as tryosine esters, but chymostatin, a chymotrypsin-specific inhibitor of actinomycetes, was not inhibitory (Takahashi et a/., 1976b). 

Phenylmethanesulfonyl fluoride (PMSF) [329-98-6] M 174.2, m 90-91 , 92-93 . Purified by recrystn from ""CgHe, pet ether or CHCl3-pet ether. [Davies and Dick J Chem Soc 483 1932 cf Tullock and Coffman J Org Chem 23 2016 I 960.] It is a general protease inhibitor (specific for trypsin and chymotrypsin) and is a good substitute for diisopropylphosphoro floridate [Fahrney and Gould 7 Am Chem Soc 85 997 1963]. 

Even though these enzymes have no absolute specificity, many of them show a preference for a particular side chain before the scissile bond as seen from the amino end of the polypeptide chain. The preference of chymotrypsin to cleave after large aromatic side chains and of trypsin to cleave after Lys or Arg side chains is exploited when these enzymes are used to produce peptides suitable for amino acid sequence determination and fingerprinting. In each case, the preferred side chain is oriented so as to fit into a pocket of the enzyme called the specificity pocket. 

Figure 11.9 A diagram of the active site of chymotrypsin with a bound inhibitor, Ac-Pro-Ala-Pro-Tyr-COOH. The diagram illustrates how this inhibitor binds in relation to the catalytic triad, the strbstrate specificity pocket, the oxyanion hole and the nonspecific substrate binding region. The Inhibitor is ted. Hydrogen bonds between Inhibitor and enzyme are striped. (Adapted from M.N.G. James et al., /. Mol. Biol. 144 43-88, 1980.)...

A closer examination of these essential residues, including the catalytic triad, reveals that they are all part of the same two loop regions in the two domains (Figure 11.10). The domains are oriented so that the ends of the two barrels that contain the Greek key crossover connection (described in Chapter 5) between p strands 3 and 4 face each other along the active site. The essential residues in the active site are in these two crossover connections and in the adjacent hairpin loops between p strands 5 and 6. Most of these essential residues are conserved between different members of the chymotrypsin superfamily. They are, of course, surrounded by other parts of the polypeptide chains, which provide minor modifications of the active site, specific for each particular serine proteinase. 

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... 

Figure 11.10 Topological diagram of the two domains of chymotrypsin, illustrating that the essential active-site residues are part of the same two loop regions (3-4 and 5-6, red) of the two domains. These residues form the catalytic triad, the oxyanion hole (green), and the substrate binding regions (yellow and blue) including essential residues in the specificity pocket.

Residue 189 is at the bottom of the specificity pocket. In trypsin the Asp residue at this position interacts with the positively charged side chains Lys or Arg of a substrate. This accounts for the preference of trypsin to cleave adjacent to these residues. In chymotrypsin there is a Ser residue at position 189, which does not interfere with the binding of the substrate. Bulky aromatic groups are therefore preferred by chymotrypsin since such side chains fill up the mainly hydrophobic specificity pocket. It has now become clear, however, from site-directed mutagenesis experiments that this simple picture does not tell the whole story. 

Figure 11.11 Schematic diagrams of the specificity pockets of chymotrypsin, trypsin and elastase, illustrating the preference for a side chain adjacent to the scisslle bond In polypeptide substrates. Chymotrypsin prefers aromatic side chains and trypsin prefers positively charged side chains that can interact with Asp 189 at the bottom of the specificity pocket. The pocket is blocked in elastase, which therefore prefers small uncharged side chains.

Figure 11.14 Schematic diagram of the active site of subtilisin. A region (residues 42-45) of a bound polypeptide inhibitor, eglin, is shown in red. The four essential features of the active site— the catalytic triad, the oxyanion hole, the specificity pocket, and the region for nonspecific binding of substrate—are highlighted in yellow. Important hydrogen bonds between enzyme and inhibitor are striped. This figure should be compared to Figure 11.9, which shows the same features for chymotrypsin. (Adapted from W. Bode et al., EMBO /.

Serine proteinases such as chymotrypsin and subtilisin catalyze the cleavage of peptide bonds. Four features essential for catalysis are present in the three-dimensional structures of all serine proteinases a catalytic triad, an oxyanion binding site, a substrate specificity pocket, and a nonspecific binding site for polypeptide substrates. These four features, in a very similar arrangement, are present in both chymotrypsin and subtilisin even though they are achieved in the two enzymes in completely different ways by quite different three-dimensional structures. Chymotrypsin is built up from two p-barrel domains, whereas the subtilisin structure is of the a/p type. These two enzymes provide an example of convergent evolution where completely different loop regions, attached to different framework structures, form similar active sites. 

Chymotrypsin shows a strong preference for hydrolyzing peptide bonds formed by the carboxyl groups of the aromatic amino acids, phenylalanine, tyrosine, and tryptophan. Flowever, over time chymotrypsin also hydrolyzes amide bonds involving amino acids other than Phe, Tyr, or Trp. Peptide bonds having leucine-donated carboxyls become particularly susceptible. Thus, the specificity... 

Trypsin, chymotrypsin, and elastase all carry out the same reaction—the cleavage of a peptide chain—and although their structures and mechanisms are quite similar, they display very different specificities. Trypsin cleaves peptides... 

Tosyl-L-phenylalanine chloromethyl ketone (TPCK) specifically inhibits chymotrypsin by covalently labeling His ". 

Partial hydrolysis of a peptide can be carried out either chemically with aqueous acid or enzymatically. Acidic hydrolysis is unselective and leads to a more or less random mixture of small fragments, but enzymatic hydrolysis is quite specific. The enzyme trypsin, for instance, catalyzes hydrolysis of peptides only at the carboxyl side of the basic amino acids arginine and lysine chymotrypsin cleaves only at the carboxyl side of the aryl-substituted amino acids phenylalanine, tyrosine, and tryptophan. 

The actual reaction mechanism is very similar for the different members of the family, but the specificity toward the different side chain, R, differs most strikingly. For example, trypsin cleaves bonds only after positively charged Lys or Arg residues, while chymotrypsin cleaves bonds after large hydrophobic residues. The specificity of serine proteases is usually designated by labeling the residues relative to the peptide bond that is being cleaved, using the notation... 

Transition state theory, 46,208 Transmission factor, 42,44-46,45 Triosephosphate isomerase, 210 Trypsin, 170. See also Trypsin enzyme family active site of, 181 activity of, steric effects on, 210 potential surfaces for, 180 Ser 195-His 57 proton transfer in, 146, 147 specificity of, 171 transition state of, 226 Trypsin enzyme family, catalysis of amide hydrolysis, 170-171. See also Chymotrypsin Elastase Thrombin Trypsin Plasmin Tryptophan, structure of, 110... 

Besides this problem of designing conformationally restricted analogs for highly specific enzymes, there are Other problems to be considered in dealing with less specific enzymes. These are discussed later in the section on locked a-chymotrypsin substrates. 

Use of conformationally restricted substrate analogs for investigating the substrate specificity of a-chymotrypsin provides an instructive example of the difficulties encountered in interpreting the results of such experiments, difficulties which, as we shall see, are especially severe for relatively nonspecific enzymes. 

There are two main classes of proteolytic digestive enzymes (proteases), with different specificities for the amino acids forming the peptide bond to be hydrolyzed. Endopeptidases hydrolyze peptide bonds between specific amino acids throughout the molecule. They are the first enzymes to act, yielding a larger number of smaller fragments, eg, pepsin in the gastric juice and trypsin, chymotrypsin, and elastase secreted into the small intestine by the pancreas. Exopeptidases catalyze the hydrolysis of peptide bonds, one at a time, fi"om the ends of polypeptides. Carboxypeptidases, secreted in the pancreatic juice, release amino acids from rhe free carboxyl terminal, and aminopeptidases, secreted by the intestinal mucosal cells, release amino acids from the amino terminal. Dipeptides, which are not substrates for exopeptidases, are hydrolyzed in the brush border of intestinal mucosal cells by dipeptidases. 

The proteases are secreted as inactive zymogens the active site of the enzyme is masked by a small region of its peptide chain, which is removed by hydrolysis of a specific peptide bond. Pepsinogen is activated to pepsin by gastric acid and by activated pepsin (autocatalysis). In the small intestine, trypsinogen, the precursor of trypsin, is activated by enteropeptidase, which is secreted by the duodenal epithelial cells trypsin can then activate chymotrypsinogen to chymotrypsin, proelas-tase to elastase, procarboxypeptidase to carboxypepti-dase, and proaminopeptidase to aminopeptidase. 

Organic solvent can affect the enzyme specificity [76]. Authors have indicated that transesterification of l,4-butyloxy-2-octylbenzene and butanol in presence of lipases from Pseudomonas can produce two different products when using hydrophilic (acetonitrile) or hydrophobic (toluene) solvents. Zaks and Klibanov [16], demonstrated that subtilisine and a-chymotrypsine specificites can be changed as a function of solvent types. This is true for a limited number of biocatalysts. 

Serine proteases usually show primary specificity (occupation of subsite Si) for positively charged arginine or lysine (trypsin, plasmin, plasminogen activators, thrombin), large hydrophobic side chains of phenylalanine, tyrosine, and tryptophan (chymotrypsin, cathepsin G, chymase, and subtilisin), or small aliphatic side chains (elastases). Nevertheless, there are a large number of variations and in many cases, other subsites like S2 and S3 are more discriminating while maintaining the... 

The 6-chloromethyl substituent (series 5 and 6) is required for the inactivation of a-chymotrypsin. Nevertheless, there is only a transient inactivation of HLE and thrombin through the formation of a stable acyl-enzyme in spite of the presence of this group as demonstrated by the spontaneous or hydroxylamine-accelerated reactivation of the treated enzymes (Scheme 11.3, pathway b).21 HLE is specifically inhibited when such an alkylating function is absent (series 7), always through the formation of a transient acyl-enzyme (Table 11.2). 

Finally, coumarin derivatives may act as general inhibitors of serine proteases or as specific inhibitors of human leukocyte elastase, depending on the nature of the substituents, through two distinct mechanisms, suicide substrates (a-chymotrypsin)... 

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