Chymotrypsin evolution

All the four essential features of the active site of chymotrypsin are thus also present in subtilisin. Furthermore, these features are spatially arranged in the same way in the two enzymes, even though different framework structures bring different loop regions into position in the active site. This is a classical example of convergent evolution at the molecular level. 

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. 

McLachlan, A.D. Gene duplications in the structural evolution of chymotrypsin. J. Mol. Biol. 128 49-79,... 

The mammalian serine proteases appear to represent a classic case of divergent evolution. All were presumably derived from a common ancestral serine protease.23 Proteins derived from a common ancestor are said to be homologous. Some nonmammalian serine proteases are 20 to 50% identical in sequence with their mammalian counterparts. The crystal structure of the elastase-like protease from Streptomyces griseus has two-thirds of the residues in a conformation similar to those in the mammalian enzymes, despite having only 186 amino acids in its sequence, compared with 245 in a-chymotrypsin. The bacterial enzymes and the pancreatic ones have probably evolved from a common precursor. 

Although the comparisons of protein folds can yield valuable insights into protein function and evolution, it is also very desirable to be able to detect structural similarities at the residue or atomic level. This is because the detection of similar patterns of functional groups in different proteins may allow analogies to be drawn between disparate proteins modes of action. The classic example of this are the similar clusters of three catalytic residues in the otherwise unrelated subtilisin and chymotrypsin families of enzymes [82]. 

The charge relay system is found at the active site of a group of enzymes called serine proteases. They include chymotrypsin, trypsin, a-lytic protease, elastase, and subtilisin. It is interesting that the charge relay system was found in enzymes belonging to different branches of diemical evolution (chymotrypsin and subtilisin). This suggests that this system is a hydrolytic catalytic system of general importance which is derived solely from amino acid residues. 

The strategy used by the cysteine proteases is most similar to that used by the chymotrypsin family. In these enzymes, a cysteine residue, activated by a histidine residue, plays the role of the nucleophile that attacks the peptide bond (see Figure 9.18). in a manner quite analogous to that of the serine residue in serine proteases. An ideal example of these proteins is papain, an enzyme purified from the fruit of the papaya. Mammalian proteases homologous to papain have been discovered, most notably the cathepsins, proteins having a role in the immune and other systems. The cysteine-based active site arose independently at least twice in the course of evolution the caspases, enzymes that play a major role in apoptosis (Section 2.4.3). have active sites similar to that of papain, but their overall structures are unrelated. 

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. 

Molecular mechanics minimization and molecular dynamics were chosen to examine the possible conformations for the two acyl enzymes and conclusions were drawn from the time evolution of the two systems. The starting point was a crystal structure of phenylethaneboronic acid bound to alpha-chymotrypsin. QUANTA/CHARMM (Brooks et al, 1983) was employed for the calculations. Ninety-five water molecules from the X-ray structure were included. Distance monitoring and the creation ofH-bonds were the main criteria for differentiating between the two molecules. Both acyl enzymes have their ketone carbonyls H-bonded to Gly-216 NH. Both start with their ester carbonyl in the oxyanion hole (H-bonded to Ser-195 and to Gly-193). The R-acyl enzyme looses both of these hydrogen bonds during the simulation. Attack of water on the R-species should, thus, be less frequently successful. Values for differences in energy were not used because of a small... 

Figure 6.17 Convergent evolution of protease active sites. The relative positions of the three key residues shown are nearly identical in the active sites of the serine proteases chymotrypsin and subtilisin.

Other enzymes that are not homologs of chymotrypsin have been found to contain very similar active sites. As noted in Chapter 6, the presence of very active sites in these different protein families is a consequence of convergent evolution. Subtilisin, a protease in bacteria such as Bacillus amyloliq-uejadens, is a particularly well characterized example. The active site of this enzyme includes both the catalytic triad and thcoxyanion hole. However, one NH groups that forms the oxyanion hole comes from the side chain of an asparagine residue rather than from the peptide backbone (Figure 9.14). 

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. 

Subtilisin (see) from Bacillus subiilis is a S.p. it resembles chymotrypsin in the hydrogen bonding of the charge-relay system, but is otherwise structurally dissimilar it is thus an example of convergent evolution of a catalytic center in two different groups of proteins. 

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