Subtilisin catalytic triad

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 /.

By changing Ser 221 in subtilisin to Ala the reaction rate (both kcat and kcat/Km) is reduced by a factor of about 10 compared with the wild-type enzyme. The Km value and, by inference, the initial binding of substrate are essentially unchanged. This mutation prevents formation of the covalent bond with the substrate and therefore abolishes the reaction mechanism outlined in Figure 11.5. When the Ser 221 to Ala mutant is further mutated by changes of His 64 to Ala or Asp 32 to Ala or both, as expected there is no effect on the catalytic reaction rate, since the reaction mechanism that involves the catalytic triad is no longer in operation. However, the enzyme still has an appreciable catalytic effect peptide hydrolysis is still about 10 -10 times the nonenzymatic rate. Whatever the reaction mechanism... 

Figure 11.16 Substrate-assisted catalysis. Schematic diagram from model building of a substrate, NHa-Phe-Ala-His-Tyr-Gly-COOH (red), bound to the subtilisin mutant His 64-Ala. The diagram illustrates that the His residue of the substrate can occupy roughly the same position in this mutant as His 64 in wild-type subtilisin (see Figure 11.14) and thereby partly restore the catalytic triad.

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. 

A structural anomaly in subtilisin has functional consequences Transition-state stabilization in subtilisin is dissected by protein engineering Catalysis occurs without a catalytic triad Substrate molecules provide catalytic groups in substrate-assisted catalysis Conclusion Selected readings... 

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... 

The molecular weight of these enzymes is around 27,000 g/mol. The active site where the catalysis takes place consists of a catalytic triad of Serine-221, Histidine-64, and Aspartate-32 (the numbers indicates the position of the amino acid in the peptide chain). A model of a subtilisin showing the binding cleft and the amino acids of the catalytic triad is illustrated through Figure 1. 

Figure 1. A model of a subtilisin showing the binding cleft and the amino acids of the catalytic triad (Serine-221, Histidine-64, and Aspartate-32)...

Mammalian PCs, just like kexin, cleave their substrates carboxy-terminal of paired basic residues and they share a conserved catalytic domain resembling that of bacterial subtilisins. The catalytically important residues Asp, His, and Ser are arranged in the catalytic triad in a way that is typical for subtilisins but distinct from the arrangement found within the (chymo)trypsin clan of serine proteases. The subtilisins and (chymo)trypsins have thus served as a prime example of convergent evolution [140,141],... 

The first crystal structure of a bacterial serine protease to be solved—subtilisin, from Bacillus amyloliquefaciens—revealed an enzyme of apparently totally different construction from the mammalian serine proteases (Figure 1.17). This was not unexpected, since there is no sequence homology between them. But closer examination shows that they are functionally identical in terms of substrate binding and catalysis. Subtilisin has the same catalytic triad, the same system of hydrogen bonds for binding the carbonyl oxygen and the acetamido NH of the substrate, and the same series of subsites for binding the acyl portion of... 

The crystal structure of subtilisin BPN dispelled this uncertainty. As already mentioned, the subtilisins and the pancreatic enzymes are dissimilar in amino acid sequence, and they proved to be dissimilar in their gross three-dimensional structure. However, the components of their catalytic site do not differ. Both enzyme groups have the same catalytic triad with hydrogen bonds linking serine to N-3 of histidine and N-1 of histidine to a buried side chain of aspartic acid (29). Since the two enzyme groups are products of different evolutionary pathways, it follows almost inescapably that this striking homology is dictated by necessity and that the buried aspartic acid is essential for catalysis. 

Figure 2 Subtilisin (PDB 1SCN) presents an identical catalytic triad to that observed in other serine proteases and enzymes yet within an entirely different protein fold.

Yet another example of the catalytic triad has been found in carboxypeptidase II from wheat. The structure of this enzyme is not significantly similar to either chymotrypsin or subtilisin (Figure 9.15). This protein is a member of an intriguing family of homologous proteins that includes esterases such as acetylcholine esterase and certain lipases. These enzymes all make use of histidine-activated nucleophiles, but the nucleophiles may be cysteine rather than serine. 

The techniques of molecular biology discussed in Chapter 6 have permitted detailed examination of the catalytic triad. In particular, site-directed mutagenesis has been used to test the contribution of individual amino acid residues to the catalytic power of an enzyme. Subtilisin has been extensively studied by this method. Each of the residues within the catalytic triad, consisting of aspartic acid 32, histidine 64, and serine 221, has been individually converted into alanine, and the ability of each mutant enzyme to cleave a model substrate has been examined (Figure 9.16). As expected, the conversion of active-site serine 221 into alanine dramatically reduced catalytic power the value of k fell to less than one-millionth of its value for the wild-type enzyme. The value of. Sf was essentially unchanged its increase by no more than a factor of two indicated that substrate binding is not significantly affected. The mutation of histidine 64 to alanine... 

Figure 9.14. The Catalytic Triad and Oxyanion Hole of Subtilisin. The peptide bond attacked by nucleophilic serine 221 of the catalytic triad will develop a negative charge, which is stabilized by enzyme NH groups (both in the backbone and in the side chain of Asn 155) located in the oxyanion hole.

Figure 9.16. Site-Directed Mutagenesis of Subtilisin. Residues of the catalytic triad were mutated to alanine, and the activity of the mutated enzyme was measured. Mutations in any component of the catalytic triad cause a dramatic loss of enzyme activity. Note that the activity is displayed on a logarithmic scale. The mutations are identified as follows the first letter is the one-letter abbreviation for the amino acid being altered the number identifies the position of the residue in the primary structure and the second letter is the one-letter abbreviation for the amino acid replacing the original one.

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. 

As opposed to the catalytic triad, which is made up of side chains that can now be mutated at will, the structure-function relationships in the oxyanion hole are not equally susceptible to experimental verification. Only in subtilisin the involvement of the side-chain amide of Asn-155 allows for quantitadve assessment of the role of the oxyanion H bonds in the stabilization of the tetrahedral intermediates. The replacement of Asn-155 with an isosteric Leu reduces the Acat by 200- to 300-fold, but leaves the Km essentially unaffected (Bryan et al., 1986). This observation is fully consistent with Asn-155 not contributing to substrate binding, but playing a key role in the stabilization of the intermediate. 

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