Catalysis catalytic triads

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... 

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. 

Catalysis, specific acid, 163 Catalytic triad, 171,173 Cavity radius, of solute, 48-49 Charge-relay mechanism, see Serine proteases, charge-relay mechanism Charging processes, in solutions, 82, 83 Chemical bonding, 1,14 Chemical bonds, see also Valence bond model... 

The mechanism for the lipase-catalyzed reaction of an acid derivative with a nucleophile (alcohol, amine, or thiol) is known as a serine hydrolase mechanism (Scheme 7.2). The active site of the enzyme is constituted by a catalytic triad (serine, aspartic, and histidine residues). The serine residue accepts the acyl group of the ester, leading to an acyl-enzyme activated intermediate. This acyl-enzyme intermediate reacts with the nucleophile, an amine or ammonia in this case, to yield the final amide product and leading to the free biocatalyst, which can enter again into the catalytic cycle. A histidine residue, activated by an aspartate side chain, is responsible for the proton transference necessary for the catalysis. Another important factor is that the oxyanion hole, formed by different residues, is able to stabilize the negatively charged oxygen present in both the transition state and the tetrahedral intermediate. 

Fructose-2,6-bisphosphatase, a regulatory enzyme of gluconeogenesis (Chapter 19), catalyzes the hydrolytic release of the phosphate on carbon 2 of fructose 2,6-bisphosphate. Figure 7-8 illustrates the roles of seven active site residues. Catalysis involves a catalytic triad of one Glu and two His residues and a covalent phos-phohistidyl intermediate. 

Zhang Y, Kua J, McCammon JA (2002) Role of the catalytic triad and oxyanion hole in acetylcholinesterase catalysis an ab initio QM/MM study. J Am Chem Soc 124 10572—10577... 

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. 

Lipases belong to the subclass of a/P-hydrolases and their structure and reaction mechanism are well understood. All lipases possess an identical catalytic triad consisting of an aspartate or glutamate, a histidine, and a nucleophilic serine residue [67], The reaction mechanism of CALB is briefly discussed as a typical example of lipase catalysis (Scheme 7). 

Carter, P., Abrahmsen, L. and Wells, J.A. (1991) Probing the mechanism and improving the rate of substrate-assisted catalysis in subtihsin BPN. Biochemistry, 30, 6142-6148. Carter, P. and Wells, J.A. (1988) Dissecting the catalytic triad of a serine protease. 

Does the "low-barrier hydrogen bond" in the catalytic triad play any special role in catalysis ... 

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... 

A similar concept was used in the development of artificial chymotrypsin mimics [54]. The esterase-site was modeled by using the phosphonate template 75 as a stable transition state analogue (Scheme 13.19). The catalytic triad of the active site of chymotrypsin - that is, serine, histidine and aspartic acid (carboxy-late anion) - was mimicked by imidazole, phenolic hydroxy and carboxyl groups, respectively. The catalytically active MIP catalyst 76 was prepared using free radical polymerization, in the presence of the phosphonate template 75, methacrylic acid, ethylene glycol dimethacrylate and AIBN. The template removal conditions had a decisive influence on the efficiency of the polymer-mediated catalysis, and best results were obtained with aqueous Na2CC>3. 

The serine proteases are a dass of proteolytic enzyme (they catalyze the hydrolysis of either ester or peptide bonds in proteins) that require an active site residue for covalent catalysis. The active site residue, the catalytic Ser-195, is particularly activated by hydrogen-bonding interactions with His-57 and Asp-102. Crystal structures show that Ser-195, His-57, and Asp-102 are dose in space. Together these three residues, which are located in the substrate binding (SI) pocket, form the famed catalytic triad of the serine proteases. In humans and mammals serine proteases perform many important functions, especially the digestion of dietary protein, in the blood-dotting cascade, and in the complement system ... 

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. 

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