Subtilisin-type proteinases

STRUCTURE OF THERMITASE, A THERMOSTABLE SERINE PROTEINASE FROM THERMOACTINOMYCES VULGARIS, AND ITS RELATIONSHIP WITH SUBTILISIN-TYPE PROTEINASES... 

Fig. 5. Phylogenetic trees of subtilisin-type proteinases. The upper part illustrates a simplified phylogenetic tree The values given correspond to calculated mutation distances [13]. The lower part shows the qualitative phylogenetic interrelationships among all the members compared.

Three-dimensional structures of four subtilisin-type enzymes, subtilisin BPN, 36373 subtilisin Carlsberg,37,383 thermitase,39,403 and proteinase K,40,413 are known, but that of aqualysin I has not yet been determined. The Ca atoms of the known structures were superimposed to obtain maximal overlap of the backbone structures, and large parts of all four structures overlap very well (Fig. 12.3) 423 On the basis of such analyses, structurally equivalent core residues (194 residues) are identified, and higher sequence identity was found to correspond to a closer overlap of mainchain atoms in the core (Table 12.1).423... 

In the family of subtilisin-type serine proteases, primary sequences of about 40 enzymes are known.423 Among them, no subtilisin produced by Bacillus species has cysteine residues. Aqualysin I (four cysteine residues per molecule),163 proteinase K (five residues),173 and thermitase (one residue)223 are cysteine-containing enzymes (Fig. 12.2). 

At present, 16 cysteine-containing subtilisin-type enzymes are known and the position of the cysteine residues is restricted to the nine corresponding sites described above.42 Of the 16 enzymes, six enzymes other than aqualysin I and proteinase K have cysteine residues at positions where the cysteine residues are able to form disulfide bond(s) like the two enzymes. Although these disulfide bonds seem to have been acquired to increase protein stability, only four kinds of disulfide bonds are found in the subtilisin-type enzymes, suggesting that the positions of the disulfide bonds have been selected strictly in the process of molecular evolution of the enzyme. 

On the basis of sequence alignment, subtilisin-type proteases can be subdivided into class I and class II.42 Subtilisins, thermitase and others, none of which has a disulfide bond, belong to class I, and ten proteases including aqualysin I and proteinase K to class II. An alkaline protease from Aspergillus oryzae, which has no cysteine residue, belongs to class II. The sequence identity between aqualysin I and the alkaline protease is 44%.49 ... 

The comparison clearly shows that thermitase is strongly homologous with subtilisins. A quantitative expression of the evolutionary relations is given by the mutation distance of proteinases of the subtilisin type [13] (Table 1). The population of the enzymes aligned in the table also involved proteinase K, a mold proteinase [6] of the subtilisin type. These data show that... 

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. 

Several proteolytic enzymes have a broad substrate specificity, but none are known which will hydrolyze all of the types of peptide bonds found in proteins. The S. griseus proteinase, papain, and the subtilisins extensively hydrolyze most proteins with liberation of free amino acids, but each enzyme also leaves many peptide bonds intact. For total enzymatic... 

EndopepUdases (proteinoses) catalyse the hydrolysis of bonds within the peptide chain, forming variously sized cleavage peptides. They can be further subdivided into acidic, neutral and basic endopeptidases. Neutral and basic types can each be divided into Serine proteases (see) and thiol proteinases (see Thiol enzymes). Examples of animal endopeptidases are Pepsin (see). Rennet enzyme (see), Ttypsin (see), Elastase (see). Thrombin (see), Plasmin (see) and Renin (see). For examples of plant and bacterial endopeptidases, see Papain, Subtilisin, Bromelain. Endopeptidases have also been isolated from yeast and fungi. 

Subtilisin (EC 3.4.21.4) an extracellular, single chain, alkaline serine protease from Bacillus subtilis and related species. S. are known from four different species of Bacillus S. Carlsberg (274 amino acid residues, M, 27,277), S. BPN (275 amino acid residues, M, 27,537), S. Novo (identical with S.BPN ) and S. amylosacchariticus (275 amino acid residues, M, 27671). The observed sequence differences between different S. represent conservative substitutions and are limited to the surface amino acids. Like the pancreatic proteinases, S. has catalytic Ser22i, His64 and Asnjj residues, but it is structurally very different from the other serine proteases, e. g. the active center of S. is -Thr-Ser-Met-, whereas that of the pancreatic enzymes is -Asp-Ser-Gly- pancreatic enzymes contain 4- disulfide bridges, whereas S. contains none S. contains 31 % a-helical structure and 3 spatially separated domains, whereas the pancreatic enzymes have 10-20% a-helical structure and a high content of p-structures in both types, the active center is a substrate cleft. S. also have a broader substrate specificity than the pancreatic enzymes. This is a notable example of the convergent evolution of catalytic activity in two structurally completely different classes of proteins. S. is used in the structural elucidation... 

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