Chymotrypsin numbering

These form the most studied class of peptidases. They have a reactive serine residue, e.g. the hydrolysis of a peptide substrate involves an acylenzyme intermediate in which the hydroxyl group of Ser195 (from the chymotrypsin numbering system) is acylated by the acyl moiety of the substrate, releasing the amine fragment of the substrate as the first product. The formation of the acylenzyme is the slow step in peptide bond hydrolysis, but the acylenzyme often accumulates in the hydrolysis of ester substrates. The acylenzyme thus formed will be the same for a series of substrates which differ in their leaving group. 

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

Numbering corresponds to chymotrypsin with active serine at 195. 

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... 

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. 

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... 

Since the imidazolide method proceeds almost quantitatively, it has been used for the synthesis of isotopically labeled esters (see also Section 3.2), and it is always useful for the esterification of sensitive carboxylic acids, alcohols, and phenols under mild conditions. This advantage has been utilized in biochemistry for the study of transacylating enzymes. A number of enzymatic transacylations (e.g., those catalyzed by oc-chymo-trypsin) have been shown to proceed in two steps an acyl group is first transferred from the substrate to the enzyme to form an acyl enzyme, which is then deacylated in a second step. In this context it has been shown[21] that oc-chymotrypsin is rapidly and quantitatively acylated by Af-fraw.s-cinnamoylimidazole to give /ra/w-cinnamoyl-a-chymotrypsin, which can be isolated in preparative quantities and retains its enzymatic activity (see also Chapter 6). 

A large number of potential reversible protease inhibitors exist (Laskowski Kato, 1980). Protein protease inhibitors like Strepromyces Subtilisin Inhibitor (SSI) (Hiromi et al, 1985) and Chymotrypsin Inhibitor (CI-2) (Jonassen, 1980 and McPhalen James, 1988) are known to be very strong inhibitors with inhibition constants at or below 10"10 M. 

Calf serum was added to the medium for cell culture to 10% concentration. T98G or U937 cells were seeded onto 25 cm2 flasks for cell culture (Nunc, Denmark). Cells were cultivated for 72 h without serum, with 10% of intact serum or 10% serum after 6 h of photodynamic treatment. After the incubation period cells were harvested with chymotrypsine and their number was calculated in a hemocy-tometer chamber. 

Proteins can be classed into groups based on their overall 3-D shapes, known as protein folds (O Figure 22-la). In general, proteins that have similar functions have similar folds. This means that if you are the proud parent of an unknown protein whose structure is solved, it may be possible to make educated guesses as to the function of the protein based on its overall fold. There are a number of well-known exceptions to this [notably, the serine protease family, subtilisin and trypsin/chymotrypsin (Hartley, 1979)], but the... 

In the late 1950s it was shown that imidazole catalyzes the hydrolyses of />-nitrophenyl acetate (7, 76) and that histidine was at the active site of a-chymotrypsin (2). These findings led Katchalski ei al. (39) to synthesize a number of histidine-containing polymers for evaluation as catalysts. Second-order rate constants were calculated on the basis of the concentration of neutral imidazole, that is, k2 = (A bs — .)/a[IM], where k , is the rate constant in the absence of catalyst and a is the fraction ionized. Some of these rate constants appear in Table I. All of the polymers possess less than... 

A number of 2-alkyl-1,2,3-benzotriazinium salts similar to those described earlier have b n used in protective coatings and in the formulation of adhesives. 3-Alkyl-l,2,3-benzotriazin-4(3fl)-ones have been examined as potential irreversible inhibitors of chymotrypsin, and the 3-(l-adamantyl) derivative as a potential virus inhibitor. Some unspecified 1,2,3-benzotriazine derivatives have been tested as radioprotectant compounds (to complement the well-known mercap-toethylamine and mercaptoalkylisothiouronium compounds), but were found to be ineffective. ... 

Multipoint attachment to a support protects the enzyme from inactivation by organic solvents. Mozhaev et al. (1990) have recently demonstrated that covalent linkage to polyacrylamide gel stabilizes df-chymotrypsin from denatmation by alcohols, the stabilizing effect increasing with the number of bonds between the protein and the support. 

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