Chymotrypsin homologous sequences

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. 

A second elastolytic enzyme (Af, 21,900) has been isolated from porcine pancreas. It shows higher activity than chymotrypsin in the hydrolysis of acetyltyrosine ester, which is used routinely to assay chymotrypsin. Another E,-like enzyme, a-lytic proteinase (Af, 19,900, 198 amino acids) has been isolated from the soil bacterium Myxobacter 495. This enzyme is remarkably similar to pancreatic E. both in structure (41 % homology, sequence in the active center Gly-Asp-Ser-Gly, 3 homologous disulfide bridges) and substrate specificity. Another E. (Af, 22,300) has been isolated from Pseudomonas aeruginosa. 

Sequences have been determined for plasminogen and bovine Factor XII, and they are not homologous with the other serine proteases. The amino-terminal sequence of Factor XII is homologous, however, with the active site of several naturally occurring protease inhibitors (11). Numbering corresponds to chymotrypsin with active serine at 195. 

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. 

For instance, the mammalian serine proteases — trypsin, chymotrypsin, and elastase—are very similar in structure and conformation. If a new mammalian serine protease is discovered, and sequence homology with known proteases... 

Fig. 2. Mapping of conserved features onto three-dimensional structure. The sequence differences between chymotrypsin and its closest homologs are mapped onto a surface representation of the structure of chymotrypsin (PDB lab9). Apeptide ligand (TPGVY) is show in stick format.

Sequence 1, in Table I, is the sequence of trypsin, chymotrypsin, pancreatic elastase, thrombin, and other mammalian proteases, and it occurs throughout the animal kingdom down to invertebrates as primitive as the sea anemone (4). The Streptomyces griseus enzymes are from Pronase, a commercial enzyme preparation. Two of its components, Streptomyces griseus trypsin and protease A, not only have the Asp.Ser.Gly sequence, but they show several other homologies in sequence with the mammalian enzymes (5, 6), The same is true for the sequence of a-lytic protease, the Myxobacter 495 enzyme (7). 

The amino acid sequence of the enzyme is homologous with those of the pancreatic enzymes and has been shown by model building to be compatible with a chymotrypsin-like three-dimensional structure and catalytic site (36). The homology in sequence is particularly well marked around His-57. The enzyme s catalytic properties are virtually indistinguishable from those of pancreatic elastase. 

The evolutionary pathways leading to such a scheme are, in part, reflected in the apparent structural homology which exists between these proteases. Sequence information points to a close relationship between trypsin and chymotrypsin (8), and there are marked similarities in the structures of carboxypeptidases A and B, both to each other and to pepsin. Such relationships seem to exist but are beyond the scope of this article. 

Considerable homology exists within the binding sites of several of the inhibitors as shown in Table VII. It has been suggested that trypsin inhibitors require a peptide sequence of Lys-X or Arg-X located within a loop of the protein closed by a disulfide bond (95,96). Reduction of disulfide bonds are known to be quite effective in destroying the inhibitory activity (77). For example, activity of the three isoinhibitors from Brazilian pink beans against both trypsin and chymotrypsin was lost when a specific disulfide bond, of the 18-21 disulfide bonds present, was reduced (77) SER appears to be a requirement of the binding site also for lysine-type inhibitors (Table VII). 

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