Chymotrypsin active site structure

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. 

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). 

Other proteases employ the same catalytic strategy. Some of these proteases, such as trypsin and elastase, are homologs of chymotrypsin. In other proteases, such as subtilisin, a very similar catalytic triad has arisen by convergent evolution. Active-site structures that differ from the catalytic triad are present in a number of other classes of proteases. These classes employ a range of catalytic strategies but, in each case, a nucleophile is generated that is sufficiently powerful to attack the peptide carbonyl group. In some enzymes, the nucleophile is derived from a side chain whereas, in others, an activated water molecule attacks the peptide carbonyl directly. 

This Highlight is part of an extraordinary story (also a cautionary tale) in the area of biocatalysis. The point of particular interest was the incredible catalytic activity claimed for so-called pepzymes - small synthetic peptides modelled to mimic the active site structures of trypsin and chymotrypsin. One was claimed to hydrolyse a simple peptide (a trypsin substrate) with efficiency comparable to that of the native enzyme. This extraordinary result provoked at least as much scepticism as excitement, and in the following months several groups tried to reproduce the results. They failed, comprehensively. [1,2] Some reasons why this failure came as no surprise were subsequently summarised by Matthews, Craik and Neurath, [3] and by Corey and Corey [4]. The background has been discussed in an Angewandte Review on Enzyme Mechanisms, Models and Mimics. [5]... 

CPA seems to occur only in mammals, but it should be noted that there is a related Zn endopeptidase, ther-molysin (EC 3.4.24.4), in thermophilic bacterium Bacillus thermoproteolyticus. Although its amino acid sequence and three-dimensional structure are unrelated to CPA. the active site structure is similar, and the mechanism of action also seems to be similar.This is an example of convergent evolution just like the case of serine proteases mammalian chymotrypsin and microbial subtilisin. 

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.)...

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... 

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... 

Steitz TA, Henderson R, Blow DM. 1969. Structure of crystalline a-chymotrypsin. 3. Crystallographic studies of substartes and inhibitors bound to the active site of a-chymotrypsin. J Mol Biol 46 337-348. 

Figure 4.3 Structure of the oxyanion hole of the active site of chymotrypsin complexed with a peptide inhibitor (PDB ICCD) (the same view as used in Figure 4.2). As discussed in the text, it is believed [43] that the oxygen atom in the oxyanion hole is protonated and therefore neutral.

The influence of the structure of the main chain on the chymotrypsin catalyzed release of p-nitroaniline is shown in Fig. 15. Oligopeptide p-nitroanili-des were attached to HPMA copolymers (as side-chains) and to polyethylene glycol (end-point attachment). From the values of kcat/KM it is evident that all oligopeptide p-nitroanilides attached to PEG were cleaved faster than those attached to HPMA copolymers. It appeared that the PEG substrates fit better into the active site of chymotrypsin due to the linearity and flexibility of the PEG molecule, and the type of spacer attachments [255]. 

Bovine pancreatic chymotrypsin (Mr 25,191) is a protease, an enzyme that catalyzes the hydrolytic cleavage of peptide bonds. This protease is specific for peptide bonds adjacent to aromatic amino acid residues (Trp, Phe, Tyr). The three-dimensional structure of chymotrypsin is shown in Figure 6-18, with functional groups in the active site emphasized. The reaction catalyzed by this enzyme illustrates the principle of transition-state stabilization and also provides a classic example of general acid-base catalysis and covalent catalysis. 

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