Chymotrypsin substrate specificity

Figure 14.4.3.2. The dependence of (A) subtilisin Carlsberg and (B) a-chymotrypsin substrate specificity for substrates 1 and 2 on the ratio of their Raoult s law activity coefficients. For the stractures of the substrates 1 and 2, and the solvents a through m in (A) and a to g in (B), refer to Ref 16. [Adapted, by permission, Ifom C.R. Wescott and A.M. Klibanov, Biotechnol. Bioeng., 56, 343(1997)].

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. 

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. 

PK Banerjee, GL Amidon. Physicochemical Property modification strategies based on enzyme substrate specificities II Alpha-chymotrypsin hydrolysis of aspirin derivatives. J Pharm Sci 70 1304, 1981. 

This endopeptidase [EC 3.4.21.20], a member of the peptidase family SI, has substrate specificity similar to that of chymotrypsin C. 

Based on the known substrate specificity of a-chymotrypsin, phenylalanine has been chosen as the amino acid at the Pi position (P-nomenclature according to Schechter and Berger) [55]. The a-proton at Pi has been substituted either by methyl or trifluoromethyl. Substitutions beyond Pi contain trifluoromethyl alanine or aminoisobutyric acid. Therefore, each fluorosubstitution can be compared to its natural as well as fluorine-free a-substituted analog, thereby enabling differentiation of the steric and electronic effects. Scheme 2 summarizes the amino acids that have been used in this study. 

Substrate specificity. Like most other enzymes, proteases display distinct preferences for certain substrates. These are often discussed using the nomenclature of Fig. 12-14. The substrate residue contributing the carbonyl of the amide group to be cleaved is designated Pj and residues toward the N terminus as P2, P3, etc., as is shown in Fig. 12-14. Residues toward the C terminus from the peptide linkage to be cleaved are designated P P etc. Chymotrypsin acts... 

Proteasomes of Thermoplasma contain a single type of p subunit but eukaryotic proteasomes contain subunits with at least three distinct substrate preferences.347 M9c They all appear to use the same hydrolytic mechanism but in their substrate specificities they are chymotrypsin-like, peptidylglutamyl-peptide hydrolyzing, branched chain amino acid preferring, and small neutral amino acid preferring based on the P, amino acid residue. In the spleen some of the P subunits of the proteasomes appear to have been replaced by proteins encoded by the major histocompatibility complex of the immune system (Chapter 31).347 This may alter the properties of the proteasome to favor their function in antigen processing. Proteasomes are also ATP- and ubiquitin-dependent, as discussed in Section 6. 

In this assay, enzymes with chymotrypsin-like specificity readily bind the side chain of Phe at the primary substrate binding site (SI) and subsequently hydrolyze the adjacent amide bond linking the Phe residue to the p-NA moiety. On cleavage, the release of p-NA is measured by the increase in absorbance at 410 nm (e4l0 = 8480 M 1 cm-1) with time, using a recording spectrophotometer. 

Hedstrpm, L., Perona, J.J., and Rutter, W.J. 1994. Converting trypsin to chymotrypsin Residue 172 is a substrate specificity determinant. Biochemistry 33 8757-8763. 

The crystal structures also provided an explanation for the different substrate specificities of trypsin, chymotrypsin,... 

L. Hedstrom, J. J. Perona, and W. J. Rutter, Converting Trypsin to Chymotrypsin Residue 172 Is a Substrate Specificity Determinant, Biochemistry, 1994a, 33, 8757-63. 

Screen, S. E., and St. Leger, R. J. (2000). Cloning, expression, and substrate specificity of a fungal chymotrypsin. Journal of Biological Chemistry, 275, 6689-6694. 

The substrate specificity of an enzyme is determined by the properties and spatial arrangement of the amino acid residues forming the active site. The serine proteases trypsin, chymotrypsin and elastase cleave peptide bonds in protein substrates on the carboxyl side of positively charged, aromatic and small side-chain amino acid residues, respectively, due to complementary residues in their active sites. 

The properties and spatial arrangement of the amino acid residues forming the active site of an enzyme will determine which molecules can bind and be substrates for that enzyme. Substrate specificity is often determined by changes in relatively few amino acids in the active site. This is clearly seen in the three digestive enzymes trypsin, chymotrypsin and elastase (see Topic C5). These three enzymes belong to a family of enzymes called the serine proteases - serine because they have a serine residue in the active site that is critically involved in catalysis and proteases because they catalyze the hydrolysis of peptide bonds in proteins. The three enzymes cleave peptide bonds in protein substrates on the carboxyl side of certain amino acid residues. 

Trypsin, chymotrypsin, and elastase are three of the most important protein-digesting enzymes secreted by the pancreas. Despite their similarities they have different substrate specificity, that is, they cleave different peptide bonds during protein digestion. 

Trypsin and factor Xa (fXa) are two members of the chymotrypsin family that have 38% sequence identity on the amino acid level and have distinguishable substrate specificities. Recently, the N-terminal 13-barrel of fXa and the C-terminal /3-barrel of trypsin were fused at a rationally designed site in the linker region between the two domains in order to create a hybrid fXa-trypsin protease (Hopfner et al., 1998). The fXa-trypsin hybrid was highly active and more active than either parent on three of the ten substrates assayed, as determined by k /Km. For most substrates, the activity of fXa-trypsin was an admixture of the two parents, probably because trypsin had higher activity than fXa for all the substrates tested. 

Perona, J. J., and Craik, C. S. (1997). Evolutionary divergence of substrate specificity within the chymotrypsin-like serine protease fold. J. Biol. Chem., 272, 29987-29990. 

Within each protease family, individual members will differ in their substrate specificity. Most proteases have extended substrate binding sites and will bind to and recognize several amino acid residues of a polypeptide substrate (see Figure 2). Usually one of these will be the primary binding site. For example, in the serine proteases chymotrypsin, trypsin, and elastase, the primary substrate binding site is the Si subsite... 

Trypsin cleaves a peptide bond on the C-terminal side of a basic residue such as arginine (Arg) or lysine (Lys) whereas chymotrypsin cleaves on the C-terminal side of the hydrophobic residues phenylalanine (Phe), tryptophan (Trp) or tyrosine (Tyr). Elastase cleaves on the C-terminal side of small amino acids such as alanine (Ala) and glycine (Gly). A large number of serine PI proteins have been isolated from plants (Table 13.4) and the substrate specificity of the target proteases corresponds with the inhibitory amino acid sequences (P2-P1-PT-P2 ) of the PI proteins. Thus, the double-headed trypsin- and chymotrypsin-inhibitory Bowman-Birk PI protein 1 (BBI-1) from soybean (Glycine BBI-1, Table 13.5G) has a Pl-PT sequence of Lys—Ser at the trypsin inhibitory domain I site and a PI PI sequence of Leu-Ser at the chymotrypsin inhibitory domain II site. 

Phage libraries have also been used to study the substrate specificity of enzymes by finding an improved artificial substrate. Coombs et al. (69) reported the detailed assessment of specificity for a serine protease belonging to the a-chymotrypsin family, the prostate specific antigen (PSA). They used both substrate optimization by singlepoint mutations and phage display libraries. The sequence of the 14-member substrate 10.2 (70) was used to start the iterative optimization process (Fig. 10.11) in which substitution or exchange of the PI, P2, or P2 residues increased the substrate affinity... 

The proteasome has multiple active sites. Three activities, chymotrypsin-like, tryp-sin-like and PGPH, are classified by their respective substrate specificities they all differ in the preferred amino acid in the PI position adjacent to the scissile amide bond. Proteasome inhibitors can be divided into several groups based on pharmacophores (Tab. 3.1, Fig. 3.7). 

The Gommission on Biochemical Nomenclature assigns enzyme numbers to 18 serine proteases in the 1972 edition of Enzyme Nomenclature (14). Seven are listed as having a trypsin-like specificity, i,e, their specific substrates have a positively charged lysine or arginine residue at Pi. Three are listed as having a chymotrypsin-like specificity, i.e., their specific substrates have residues of tryptophan, tyrosine, phenylalanine, or leucine at Pi, i,e, residues with bulky, hydrophobic side chains. Two enzymes have elastase-like specificities. They prefer a residue with... 

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