Chymotrypsin active sites

Search the Enzyme Structure Database for y-chymotrypsin active site (by the aid of the active-site-modified enzyme or active-site-specific inhibitor-enzyme complex) to identify and depict (save pdb file) the catalytic triad of y-chymotrypsin. 

The third approach to solving this problem (Farber, 1999) involves the preparation of an enzyme-intermediate complex at high substrate concentration for X-ray data collection. Under such a condition active sites in the crystal lattice will be filled with intermediates. Using a combination of flow cell experiments and equilibrium experiments, it is possible to obtain the structure of important intermediates in an enzyme reaction (Bolduc et al., 1995). It was also discovered that some enzyme crystals can be transformed from their aqueous crystallization buffer to nonaqueous solvents without cross-linking the crystals before the transfer (Yennawar et. al., 1995). It is then possible to regulate the water concentration in the active site. The structure of the first tetrahedral intermediate, tetrapeptide -Pro-Gly-Ala-Tyr- in the y-chymotrypsin active site obtained by this method is shown in Fig. 1.1. 

Figurel.10. Stereo diagram of the acyl moiet of the spin-labeled tryptophanyl-acylenzyme reaction intermediate of a-chymotrypsin. Active site residues close to the acyl moiety are labeled (Makinen et al., 1998). Reproduced with permission.

Fig. 2.13 illustrates the electrostatic effects in transition state in enolase reaction (Larson et al., 1996). During this reaction a proton is removed by Lys-345 from C-2 of 2-phosphoglycerate to give an enolyzed, charged intermediate. This intermediate is stabilized by electrostatic interaction with five positive charges supplied by two Mg+2 ions and a protonated lysine. The 10-11 electrostatic interactions were found in the transition state of formate dehydrogenase and carbamoyl synthetase (Bruice and Benkovic, 2000) Another example of multifunctional interactions during enzymatic reactions in intermediate is the X-ray structure of tetrahedral intermediate in the chymotrypsin active site (Fig. 1.1). 

Blevins and Tulinsky (1985) suggested two functions for the solvent at the chymotrypsin active site (1) solvation of the Asp—His—Ser catalytic triad, and (2) a guiding effect on the substrate in formation of the enzyme-substrate complex, provided by several waters at the end of the specificity site. X-Ray diffraction results have suggested a role of active-site water in determining the kinetics or equilibria of substrate binding for other proteins (Section IV). 

Figure 13 Phenyl hippurate substrate—chymotrypsin active site complex. Dashed lines show distance constraints assigned for docking.

If the comparison of Lewis acid catalysis vs general acid-base catalysis is extended to the specific examples of carboxypeptidase A and a-chymotrypsin, then it is interesting to note that the function of the carboxypeptidase active site catalytic residues (33, 34) appears to involve activation of the substrate for the chemical transformation primarily via Lewis acid catalysis (see Scheme III, Section IV). In contrast, the function of the a-chymotrypsin active site catalytic residues appears to involve the activation of the hydroxyl of Ser-195... 

Cram s group, like that of Lehn, is attempting to imitate enzyme catalysis via kinetic acceleration through host-guest complexation. A series of transacylase mimics have been synthesized [44] which contain the complexing site, the proton transfer catalyst and the nucleophile found in the chymotrypsin active site. Relatively rapid rates of acylation were observed. It is obvious, however, that additional information will have to be acquired and more model compounds synthesized before the mode of action of an enzyme such as chymotrypsin can be clarified. 

Fig. 10.12 Sequence alignment of trypsin, chymotrypsin and thrombin (bovine). The active sites histidine, aspartic acid and serine are highlighted.

Inhibitors as well as substrates bind in this crevice between the domains. From the numerous studies of different inhibitors bound to serine pro-teinases we have chosen as an illustration the binding of a small peptide inhibitor, Ac-Pro-Ala-Pro-Tyr-COOH to a bacterial chymotrypsin (Figure 11.9). The enzyme-peptide complex was formed by adding a large excess of the substrate Ac-Pro-Ala-Pro-Tyr-CO-NHz to crystals of the enzyme. The enzyme molecules within the crystals catalyze cleavage of the terminal amide group to produce the products Ac-Pro-Ala-Pro-Tyr-COOH and NHs. The ammonium ions diffuse away, but the peptide product remains bound as an inhibitor to the active site of the enzyme. 

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. 

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.

The active site of subtilisin is outside the carboxy ends of the central p strands analogous to the position of the binding sites in other a/p proteins as discussed in Chapter 4. Details of this active site are surprisingly similar to those of chymotrypsin, in spite of the completely different folds of the two enzymes (Figures 11.14 and 11.9). A catalytic triad is present that comprises residues Asp 32, His 64 and the reactive Ser 221. The negatively charged oxygen atom of the tetrahedral transition state binds in an oxyanion hole,... 

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 /.

All the four essential features of the active site of chymotrypsin are thus also present in subtilisin. Furthermore, these features are spatially arranged in the same way in the two enzymes, even though different framework structures bring different loop regions into position in the active site. This is a classical example of convergent evolution at the molecular level. 

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. 

Figure 16.21 Structure of one subunit of the core protein of Slndbls virus. The protein has a similar fold to chymotrypsin and other serine proteases, comprising two Greek key motifs separated by an active site cleft. The C-terminus of the protein is bound in the catalytic site, making the coat protein inactive (Adapted from S. Lee et al., Structure 4 531-541, 1996.)...

Plotnick, M. L, Mayne, L., Schechter, N. M., and Rnbiii, H., 1996. Distortion of the active site of chymotrypsin complexed widi a serpin. Biochemistry 35 7586-7590. 

All peptidases within a family will have a similar tertiary structure, and it is not uncommon for peptidases in one family to have a similar structure to peptidases in another family, even though there is no significant sequence similarity. Families of peptidases with similar structures and the same order of active site residues are included in the same clan. A clan name consists of two letters, the first representing the catalytic type as before, but with the extra letter P , and the second assigned sequentially. Unlike families, a clan may contain peptidases of more than one catalytic type. So far this has only been seen for peptidases with protein nucleophiles, and these clans are named with an initial P . Only three such clans are known. Clan PA includes peptidases with a chymotrypsin-like fold, which besides serine peptidases such as chymotrypsin... 

The elucidation of the X-ray structure of chymotrypsin (Ref. 1) and in a later stage of subtilisin (Ref. 2) revealed an active site with three crucial groups (Fig. 7.1)-the active serine, a neighboring histidine, and a buried aspartic acid. These three residues are frequently called the catalytic triad, and are designated here as Aspc Hisc Serc (where c indicates a catalytic residue). The identification of the location of the active-site groups and intense biochemical studies led to several mechanistic proposals for the action of serine proteases (see, for example, Refs. 1 and 2). However, it appears that without some way of translating the structural information to reaction-potential surfaces it is hard to discriminate between different alternative mechanisms. Thus it is instructive to use the procedure introduced in previous chapters and to examine the feasibility of different... 

To realize the reason for this result from a simple intuitive point of view it is important to recognize that the ionized form of Aspc is more stable in the protein-active site than in water, due to its stabilization by three hydrogen bonds (Fig. 7.7). This point is clear from the fact that the observed pKa of the acid is around 3 in chymotrypsin, while it is around 4 in solution. As the stability of the negative charge on Aspc increases, the propensity for a proton transfer from Hisc to Aspc decreases. 

Enzyme active sites, 136,148, 225. See also Protein active sites in carbonic anhydrase, 197-199 in chymotrypsin, 173 in lysozyme, 153, 157 nonpolar (hypothetical site), 211-214 SNase, 189-190,190 steric forces in, 155-158, 209-211, 225 in subtilisin, 173 viewed as super solvents, 227 Enzyme cofactors calcium ... 

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... 

In conclusion, one must be aware of these limitations on the use of locked substrate analogs. The problems encountered in the study of a-chymotrypsin are perhaps more severe than for most other enzymes, since a-chymotrypsin normally acts on large, polymeric substrates and is relatively nonspecific. The active site of a-chymotrypsin therefore potentially can bind small substrates such as D24 in a variety of ways. Ideally, larger conformationally restricted substrates should give more information about the active site of a-chymotrypsin. However, besides the increased problems involved in synthesizing these larger substrates, there is the problem of increased possibility of uncertainty in their conformations. 

Figure 9-6. Selective proteolysis and associated conformational changes form the active site of chymotrypsin, which includes the Aspl 02-His57-Ser195 catalytic triad. Successive proteolysis forms prochymotrypsin (pro-CT), Jt-chymotrypsin (jt-CT),and ultimately a-chymotrypsin (a-CT), an active protease whose three peptides remain associated by covalent inter-chain disulfide bonds.

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. 

However, diffusion of the reactive QM out of the enzyme active site is a major concern. For instance, a 2-acyloxy-5-nitrobenzylchloride does not modify any nucleophilic residue located within the enzyme active site but becomes attached to a tryptophan residue proximal to the active site of chymotrypsin or papain.23,24 The lack of inactivation could also be due to other factors the unmasked QM being poorly electrophilic, active site residues not being nucleophilic enough, or the covalent adduct being unstable. Cyclized acyloxybenzyl molecules of type a could well overcome the diffusion problem. They will retain both the electrophilic hydroxybenzyl species b, and then the tethered QM, in the active site throughout the lifetime of the acyl-enzyme (Scheme 11.1). This reasoning led us to synthesize functionalized... 

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