Catalytic triads

Figure 11.6 A schematic view of the presumed binding mode of the tetrahedral transition state intermediate for the deacylation step. The four essential features of the serine proteinases are highlighted in yellow the catalytic triad, the oxyanion hole, the specificity pocket, and the unspecific main-chain substrate binding.

The enzyme provides a general base, a His residue, that can accept the proton from the hydroxyl group of the reactive Ser thus facilitating formation of the covalent tetrahedral transition state. This His residue is part of a catalytic triad consisting of three side chains from Asp, His, and Ser, tvhich are close to each other in the active site, although they are far apart in the amino acid sequence of the polypeptide chain (Figure 11.6). 

Figure 11.7 Schematic diagram of the structure of chymotrypsin, which is folded into two antiparallel p domains. The six p strands of each domain are red, the side chains of the catalytic triad are dark blue, and the disulfide bridges that join the three polypeptide chains are marked in violet. Chain A (green, residues 1-13) is linked to chain B (blue, residues 16-146) by a disulfide bridge between Cys 1 and Cys 122. Chain B is in turn linked to chain C (yellow, residues 149-245) by a disulfide bridge between Cys 136 and Cys 201. Dotted lines indicate residues 14-15 and 147-148 in the inactive precursor, chmotrypsinogen. These residues are excised during the conversion of chymotrypsinogen to the active enzyme chymotrypsin.

The active site is situated in a crevice between the two domains. Domain 1 contributes two of the residues in the catalytic triad. His 57 and Asp 102, whereas the reactive Ser 195 is part of the second domain (see Figure 11.7). 

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. 

His 57 and Ser 195 are within loop 3-4 of domains 1 and 2, respectively. The third residue in the catalytic triad. Asp 102, is within loop 5-6 of domain 1. The rest of the active site is formed by two loop regions (3-4 and 5-6) of domain 2. As in so many other protein structures described previously, the barrels apparently provide a stable scaffold to position a few loop regions that constitute the essential features of the active site. 

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 C-terminal part is green. The catalytic triad Asp 32, His 64, and Ser 221 as well as Asn 15S, which forms part of the oxyanion hole are shown in purple. The main chain of part of a polypeptide Inhibitor is shown in red. Main-chain residues around 101 and 127 (orange circles) form the nonspecific binding regions of peptide substrates. 

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 /.

The answer is quite clear. His 64, which is part of the catalytic triad, is in the first turn of helix Ob (Figure 11.13). This helix would be on the other side of the P sheet, far removed from the active site if the motif P2-o.b-P3 were right-handed. Therefore, to produce a proper catalytic triad of Asp 32, His 64, and Ser 221, helix Ob must be on the same side of the p sheet as Ser 221 consequently, the motif has evolved to be left-handed. 

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. 

The catalytic triad consists of the side chains of Asp, His, and Ser close to each other. The Ser residue is reactive and forms a covalent bond with the substrate, thereby providing a specific pathway for the reaction. His has a dual role first, it accepts a proton from Ser to facilitate formation of the covalent bond and, second, it stabilizes the negatively charged transition state. The proton is subsequently transferred to the N atom of the leaving group. Mutations of either of these two residues decrease the catalytic rate by a factor of 10 because they abolish the specific reaction pathway. Asp, by stabilizing the positive charge of His, contributes a rate enhancement of 10. ... 

Carter, P, Wells, J.A. Dissecting the catalytic triad of a serine protease. Nature 332 564-568, 1988. 

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... 

FIGURE 16.17 Structure of chyinotrypsiu (white) ill a complex with egliii C (blue ribbon structure), a target protein. The residues of the catalytic triad (His ", Asp , and Ser ) are highlighted. His (blue) is flanked above by Aspios on the right by Ser (yel-... 

Until recently, the catalytic role of Asp ° in trypsin and the other serine proteases had been surmised on the basis of its proximity to His in structures obtained from X-ray diffraction studies, but it had never been demonstrated with certainty in physical or chemical studies. As can be seen in Figure 16.17, Asp ° is buried at the active site and is normally inaccessible to chemical modifying reagents. In 1987, however, Charles Craik, William Rutter, and their colleagues used site-directed mutagenesis (see Chapter 13) to prepare a mutant trypsin with an asparagine in place of Asp °. This mutant trypsin possessed a hydrolytic activity with ester substrates only 1/10,000 that of native trypsin, demonstrating that Asp ° is indeed essential for catalysis and that its ability to immobilize and orient His is crucial to the function of the catalytic triad. 

The metabolic breakdown of triacylglycerols begins with their hydrolysis to yield glycerol plus fatty acids. The reaction is catalyzed by a lipase, whose mechanism of action is shown in Figure 29.2. The active site of the enzyme contains a catalytic triad of aspartic acid, histidine, and serine residues, which act cooperatively to provide the necessary acid and base catalysis for the individual steps. Hydrolysis is accomplished by two sequential nucleophilic acyl substitution reactions, one that covalently binds an acyl group to the side chain -OH of a serine residue on the enzyme and a second that frees the fatty acid from the enzyme. 

Figure 29.2 MECHANISM Mechanism of action of lipase. The active site of the enzyme contains a catalytic triad of aspartic acid, histidine, and serine, which react cooperatively to carry out two nucleophilic acyl substitution reactions. Individual steps are explained in the text.

The esteratic subsite contains the catalytic machinery of the enzyme. The catalytic triad residues - Ser 200, His 440 and Glu 327 (the residue numbering in this section refers to Torpedo californica acetylcholinesterase, TcAChE) - are identical in both enzymes and basically in the same positions. 

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... 

---