Subtilisin, active serine, mechanism

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... 

An exceptionally reactive serine residue has been identified in a great number of hydrolase enzymes, e. g., trypsin, subtilisin, elastase, acetylcholine esterase and some lipases. These enzymes appear to hydrolyze their substrates by a mechanism analogous to that of chymotrypsin. Hydrolases such as papain, ficin and bromelain, which are distributed in plants, have a cysteine residue instead of an active serine residue in their active sites. Thus, the transient intermediates are thioesters. 

Convergence may also occur when the sequence and structure of molecules are very different, but the mechanisms by which they act are similar. Serine proteases have evolved independently in bacteria (e.g. subtilisin) and vertebrates (e.g. trypsin). Despite their very different sequences and three-dimensional structures, in each the same set of three amino acids form the active site. The catalytic triads are His57, Aspl02, and Serl95 (trypsin) and Asp32, His64, and Ser221 (subtilisin) (Doolittle, 1994 A. Tramontano, personal communication). 

EXAMPLE 5.20 In the catalytic mechanism of the serine protease (so called because of a catalytically important serine in the active site) subtilisin, the tetrahedral intermediate is thought to be stabilized by a hydrogen bond on the side chain of Asnl55. Replacement of Asnl55 with glycine leaves the binding unaffected, but inhibited in the catalytic step, confirming the proposed mechanism. 

CPA seems to occur only in mammals, but it should be noted that there is a related Zn endopeptidase, ther-molysin (EC 3.4.24.4), in thermophilic bacterium Bacillus thermoproteolyticus. Although its amino acid sequence and three-dimensional structure are unrelated to CPA. the active site structure is similar, and the mechanism of action also seems to be similar.This is an example of convergent evolution just like the case of serine proteases mammalian chymotrypsin and microbial subtilisin. 

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. 

Trypsin, a mammelian protease, and subtilisin, a bacterial protease, have both been shown to have a mechanism of action similar to a-chymotrypsin. While a-chymotrypsin and subtilisin have totally different foldings of their polypeptide backbones, the residues involved in catalysis (serine, histidine, aspartic acid) have the same spatial relationships. This similarity of active centers is a prime example of convergent evolution of active center geometries in enzymes (77). 

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