Trypsin catalytic triad

A four helix fold has also been used for de novo design of chymotiypsin-like enzyme (Hahn, Wieskaw and Stewart, 1990). The trypsin catalytic triad SER, HIS and ASP has... 

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. 

NS3 is a 631 amino acid protein, and its first 180 amino acids encode a serine protease of the chymotrypsin family (Figure 2.2A). It has a typical chymotrypsin-family fold consisting of two jS-barrels, with catalytic triad residues at the interface. His-57 and Asp-81 are contributed by the N-terminal jS-barrel and Ser-139 from the C-terminal jS-barrel. NS3 and closely related viral proteases are significantly smaller than other members of the chymotrypsin family, and many of the loops normally found between adjacent jS-strands in trypsin proteases are truncated in NS3 [31]. Probably... 

The outstanding inclusion ability and the carboxylic functions of host I raised the idea of co-erystallizing it with imidazole (Im) which, due to its versatile nature 114), is one of the frequently used components in enzyme active sites, generally presented by histidine. Formally, a system made of imidazole and an acid component may mimic two essential components of the so-called catalytic triad of the serine protease family of enzymes the acid function of Aspl02 and the imidazole nucleus of His57 115) (trypsin sequence numbering). The third (albeit essential) component of the triad corresponding to the alcohol function of Seri 95 was not considered in this attempt. This family of enzymes is of prime importance in metabolitic processes. 

Fig. 3.3. Major steps in the hydrolase-catalyzed hydrolysis of peptide bonds, taking chymo-trypsin, a serine hydrolase, as the example. Asp102, His57, and Ser195 represent the catalytic triad the NH groups of Ser195 and Gly193 form the oxyanion hole . Steps a-c acylation Steps d-f deacylation. A possible mechanism for peptide bond synthesis by peptidases is represented by the reverse sequence Steps f-a.

Among cysteine proteases of bacteria is a papainlike enzyme from Clostridium histolyticum with a specificity similar to that of trypsin 338 Tire anaerobic Porphyromonas gingivalis, which is implicated in perio-dental disease, produces both arginine- and lysine-specific cysteine proteases designated gingipains.339 3393 Some virally encoded cysteine proteases, including one from the polio virus, have trypsin-like sequences with the serine of the catalytic triad replaced by cysteine.340 341 A human adenovirus protease also has a Cys His Glu triad but a totally different protein fold.342... 

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],... 

Fig. 7.1 The tetramer of eco bound to a serine protease. Visualized as a cartoon of the canonical protease and eco interaction (a), and (b), as two views of the three dimensional solution of D102N trypsin in complex with eco [3]. Each eco molecule has three protein-protein interaction surfaces. The C-terminus forms an anti-parallel p ribbon to complete the ecotin dimer interface. The 80 s and 50 s loops form the primary binding site by interacting with the protease at the active site cleft in a sub-strate-like y -sheet conformation. The 60 s and lOO s loops of eco form the secondary binding site by interacting with the C-termi-nal a-helix of the protease. Note that each eco molecule contacts both of the protease molecules. Two eco molecules (black and medium grey) form a pair of interactions each with two protease molecules (light grey). The catalytic triad residues Ser-195, Asp-102 and His-57 are in black ball and stick representation. This figure was made with Molscript [37] and Raster 3D [38].

FIGURE 1. (A) Catalytic triad in the serine protease trypsin (PDB ID lAKS), wherein the nucle-ophilicity of the nucleophilic Serl95 is dramatically enhanced by H-bonding (dotted lines) with H57. (B) The metaUotriad active site in serralysin (PDB ID ISRP), showing the sandwiched nucleophihc water (in the form of hydroxide) by the active-site Zn(II) and Glul77 via a coordination bond and a H-bond... 

More than a third of all known proteolytic enzymes are serine proteases (2). The family name stems from the nucleophilic serine residue within the active site, which attacks the carbonyl moiety of the substrate peptide bond to form an acyl-enzyme intermediate. Nucleophilicity of the catalytic serine is commonly dependent on a catalytic triad of aspartic acid, histidine, and serine—commonly referred to as a charge relay system (3). First observed by Blow over 30 years ago in the structure of chymotrypsin (4), the same combination has been found in four other three-dimensional protein folds that catalyze hydrolysis of peptide bonds. Examples of these folds are observed in trypsin. 

Many other proteins have subsequently been found to contain catalytic triads similar to that discovered in chymotrypsin. Some, such as trypsin and elastase, are obvious homologs of chymotrypsin. The sequences of these proteins are approximately 40% identical with that of chymotrypsin, and their overall structures are nearly the same (Figure 9.12). These proteins operate by mechanisms identical with that of chymotrypsin. However, they have very different substrate specificities. Trypsin cleaves at the peptide bond after residues with long, positively charged side chains—namely, arginine and lysine—whereas elastase cleaves at the peptide bond after amino acids with small side chains—such as alanine and serine. Comparison of the Sj pockets of these enzymes reveals the basis of the specificity. 

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. 

Ramos et al. [238] have also used the QM/MM LSCF method [312] to understand the reasons why the pancreatic trypsin inhibitor, PTI, behaves as an inhibitor of trypsin rather than as a substrate. In fact, PTI places a peptidic bond between a lysine and an alanine in the catalytic triad of trypsin with the side chain of the lysine in the binding pocket of the enzyme, exactly as a cleavable peptide would do, and this provokes no reaction between PTI and trypsin. The QM/MM calculations performed show that the geometry adopted by the active site of the enzyme in the complex is such that it prevents the nucleophilic attack of the hydroxyl oxygen on the peptide bond of PTI. 

Corey, D. R., and Craik, C. S. An investigation into the minimum requirements for peptide hydrolysis by mutation of the catalytic triad of trypsin. J. Amer. Chem. Soc. 114, 1784-1790 (1992). 

Both AChE and BChE are of the serine hydrolase class, which includes proteases such as trypsin (see PROTEASE inhibitors). Characteristically, such enzymes can be inhibited through covalent linkage of constituent parts of irreversible anticholinesterases such as dyflos (DFP, diisopropylfluorophosphonate). The active site of the enzyme contains a catalytic triad with a glutamate residue, a serine residue and a histidine imidazole ring. The mechanism of the catalysis of break down of AChE has been characterized, and the reaction progresses at a very fast rate. 

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