Subtilisin-Like Enzymes

Like chymobypsin, subtilases can catalyze reactions leading to special peptides. Proteinase K catalyzed the polymerization of L-Phe in the presence of tris(2-aminoethyl) amine to form a branched oligo(L-phenylalanine), which showed self-assembly to form fluorescent fibers [17]. 

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Groen, H., Bech, L.M., Branner, S. andBreddam, K. (1990) A highly active and oxidation-resistant mutant subtilisin-like enzyme produced by a combination of site-directed mutagenesis and chentical modification. Eur. J. Biochem., 194, 897-901. 

Wlodawer, A., Li, M., Dauter, Z., Gustchina, A., Uchida, K., Oyama, H., Dunn, B. M., and Oda, K. (2001). Carboxyl proteinase from Pseudomonas defines a novel family of subtilisin-like enzymes. Natl. Struct. Biol., 8, 442-446. 

Furin, also known as paired basic amino-acid-cleaving enzyme (PACE), is a membrane bound subtilisin-like serine protease of the irons Golgi compartment. It is ubiquitously expressed and mediates processing of many protein precursors at Arg-X-Lys/Arg-Arg sites. 

Members of both structural families of serine proteinases, the trypsinlike and the subtilisin-like, have been found to bind Ca " (references in Tables 1 and II). The role of Ca " in all of these proteolytic enzymes appears to be one of stabilization of structure and/or maintenance of... 

St Leger et al. (1987b) characterized two subtilisin-like proteases (chymoelastases) and three tiypsin-like proteases from M. anisopliae. A subtilisin-like protease (Prl pl=10.3, Mw=25 kDa) and a trypsin-like protease (Pr2 pl=4.42, Mw=28.5 kDa) were purified to homogeneity. Inhibition studies have revealed that both enzymes possess essential Ser and His residues in the active site. Prl exhibited higher activity to locust cuticle than Pr2 and it showed activity to elastin as well. 

The greatest difference in RFLP patterns at all prl genes was observed in the case of M. anisopliae var. majus strain 473 and a M. flavoviride isolate when compared with other M anisopliae strains. The prl genes were concluded to represent a gene family of subtilisin-like proteases and prl A was supposed to encode for the ancestral enzyme which was subsequently duplicated and rearranged within the genome. 

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. 

Figure 3 Cysteine protease and subtilisin-like protease pathways for proneuropeptide processing. Distinct cysteine protease and subtilisin-like protease pathways have been demonstrated for pro-neuropeptide processing. Recent studies have identified secretory vesicle cathepsin L as an important processing enzyme for the production of the endogenous enkephalin opioid peptide. Preference of cathepsin L to cleave at the NH2-terminal side of dibasic residue processing sites yields peptide intermediates with NH2-terminal residues, which are removed by Arg/Lys aminopeptidase. The well-established subtilisin-like protease pathway involves several prohormone convertases (PC). PC1/3 and PC2 have been characterized as neuroendocrine processing proteases. The PC enzymes preferentially cleave at the COOH-terminal side of dibasic processing sites, which results in peptide intermediates with basic residue extensions at their COOH-termini that are removed by carboxypeptidase E/H.

All human metzincins are secreted as proenzymes. Astacins and adamalysins are mostly activated by calcium-ion-dependent serine proteases pro-protein convertases) that meet up with their substrates in trans-Golgi and secretory vacuoles. These proenzymes are known as furin-like convertases because of their homology to a serine protease called furin and a bacterial endoprotease called subtilisin. The furin-like enzymes require calcium ions to maintain structural stability whereas other serine proteases, represented by trypsin and chymotrypsin, do not. The furin-like pro-protein convertases autocleave their own N-terminal domain propeptide (self-activate) during secretion and then convert the N-terminal domains of co-secreted metzincins. Activation cascades also occur among the... 

Pepsin, pepsin-like enzymes, chymosin, rennin, and other acid proteinases have an activity optimum at pH 2.0-3.5 papain, trypsin, chymotrypsin, and similar enzymes are most active at neutral pH (pH 6-8). Subtilisin BPN, pancreatic elastase, leucine... 

Thus, GAP-releasing enzyme likely shares sequence homology with PC 1/3 and may represent bovine hypothalamic PCl/3. In any case, GAP-releasing enzyme should be considered a member of the subtilisin-like family of pro-hormone processing enzymes. PC 1/3 and PC2 are found in various regions of the mouse hypothalamus (36,37,44), However, PCl/3 is the more abundant enzyme. 

The closest organic specie to the inorganic boric acid are the boronic acids generally described as R-B(OH)2. Boronic acids have been shown to act as inhibitors of the subtilisins. X-ray crystallographic studies of phenylboronic acid and phenyl-ethyl-boronic acid adducts with Subtilisin Novo have shown that they contain a covalent bond between the oxygen atom of the catalytic serine of the enzyme and the inhibitor boron atom (Matthews et al, 1975 and Lindquist Terry, 1974). The boron atom is co-ordinated tetrahedrally in the enzyme inhibitor complex. It is likely that boric acid itself interacts with the active site of the subtilisins in the same manner. 

Little was done in the area of cross-linked enzyme crystals over the next 10 years. In 1977, the kinetic properties of CLCs of the protease subtilisin were reported by Tuchsen and Ottesen [3], They reported that cross-linked enzyme crystals of subtilisin were highly effective catalysts with increased thermal stability and increased stability toward acid compared to the soluble enzyme. They further reported that the CLCs of subtilisin showed essentially no autodigestion at 30°C. Like Quiocho and Richards before them, Tuchsen and Ottesen found... 

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