Elastase, catalytic triad

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. 

It is unclear, however, if the operation of the catalytic triad imparts any catalytic advantage. This was investigated in a systematic study of human leukocyte elastase [14, 42]. For p-nitroanilides of substrates R-Val-, R-Pro-Val-, R-Ala-Pro-Val-, and R-... 

Figure 6.7 Catalytic triad involved in elastase action.

Studies with ab initio types of hybrid potential include the early work of Weiner et al. on the nature of catalysis in trypsin and the studies of the catalytic activity of phospholipase A2 by Hillier et al. Investigations with semiempirical hybrid potentials are more extensive and include calculations of the reactions in triosephosphate isomerase by Bash et al. and in chorismate mutase by Lyne et al. and a study of the proton jump in the catalytic triad of human neutrophil elastase. The study of the chorismate mutase reaction was especially interesting because the enzyme is the only known one that catalyzes a pericyclic reaction that also occurs readily in solution. The results of the hybrid study were particularly lucid in this case because the enzyme works, not by chemically catalyzing the reaction, but by preferentially binding a distorted form of the substrate and stabilizing the transition state. 

Comparison (or alignment) of amino acid sequences, also called homology search, often provides first-hand information on such conserved structural features and enables one to classify enzymes into families and predict the possible function of a new enzyme (86). A family of enzymes usually folds into similar 3-D structures, at least at the active site region. A typical example is the serine protease family whose members—trypsin, chymotrypsin, elastase, and subtilisin—commonly contain three active-site residues, Asp/His/Ser, which are known as the catalytic triad or charge relay system. Another example is the conserved features of catalytic domains of the highly diverse protein kinase family. In this kinase family, the ATP-binding (or phosphate-anchoring) sites present a consensus sequence motif of Gly-X-Gly-X-X-Gly (67,87). 

Figure 25.3 shows the relationship of active site of serine hydrolases. The serine hydrolases include serine proteases, lipases, and PHB depolymerases. A common feature of the serine proteases is the presence of a specific amino acid sequence -Gly-Xl-Ser-X2-Gly-. The catalytic mechanism of these enzymes is very similar and the catalytic center consists of a triad of serine, histidine, and aspartate residues [54]. The serine from this sequence attacks the ester bond nucleophilically [55]. Lipases and PHB depolymerases also have a common amino acid sequence around the active site, -Gly-Xl-Ser-X2-Gly-. These serine hydrolases may share a similar mechanism of substrate hydrolysis [21, 56]. In terms of origin of enzymes, it would be wise to consider that the enzyme had wide substrate specificity initially, and then it started to evolve gradually for each specific substrate. In the case of polyester hydrolysis, lipases showed the widest substrate specificity among serine hydrolases for hydrolysis of various polyesters ranging from a-ester bonds to (o-ester bonds. PHB depolymerases would become more specific for microbial PHB that has / -ester bonds, though it could also hydrolyze other polyesters that have -ester and y-ester bonds. Serine proteases such as proteinase K, subtilisin, a-chymotrypsin, elastase, and trypsin hydrolyze only optically active PLLA with a-ester bonds and various proteins with a-amido bonds. 

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