Cysteine-containing subtilisin

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. 

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). 

Yet another example of the catalytic triad has been found in carboxy-peptidase II from wheat. The structure of this enzyme is not significantly similar to either chymotrypsin or subtilisin (Figure 9.15). This protein is a member of an intriguing family of homologous proteins that includes esterases such as acetylcholine esterase and certain lipases. These enzymes all make use of histidine-activated nucleophiles, but the nucleophiles may be cysteine rather than serine. Finally, other proteases have been discovered that contain an active-site serine or threonine residue that is activated not by a histidine-aspartate pair but by a primary amino group from the side chain of lysine or by the N-terminal amino group of the polypeptide chain. 

The construction of synthetic selenocysteine-containing proteins or selenium-containing proteins attracts considerable interest at present, mainly for the reason that it can be used to solve the phase problem in X-ray crystallography. Selenomethionine incorporation has been used mostly until now for this purpose. There are also two reports on new synthetic selenocysteine-containing proteins. In one case, the active site serine of subtilisin has been converted into a selenocysteine residue by chemical means, with the result that the enzyme gains a predominant esterase instead of protease activity. In the second case, automated peptide synthesis was carried out to produce a peptide in which all seven-cysteine residues of the Neurospora crassa metallothionein (Cu) were replaced by selenocysteine. The replacement resulted in an alteration of both the stoichiometry and the affinity of copper binding. ... 

The inhibitor has been purified using a modification of the scheme of Millet and Gregoire [2] (Table 2) plus a final fractionation on a reverse phase HPLC column employing an n-butanol gradient (in 0.1% trifluoroacetic acid). The amino acid composition differs somewhat from inhibitors of E. coli or Streptomyces (Table 3) but they are probably all of the same general class in that they contain a low percentage of cysteine and are active on either subtilisin (E. coli and Streptomyces) or related serine proteases [8],... 

Serine and cysteine protease-catalyzed hydrolysis of esters and amides involves two steps (1) attack of the active site nucleophile on the carbonyl at the cleavage site to form an acyl-enzyme intermediate, and (2) hydrolysis of the acylated enzyme to regenerate the catalyst (Equation 1). We wondered whether a selenol group (-SeH) could assume the role of the active site nucleophile. We found that selenolsubtilisin, like thiolsubtilisin, is a poor amidase. Thus, i F-succinyl-Ala-Ala-Pro-Phe-p-nitro-anilide is not hydrolyzed at all by the selenol-containing enzyme even though it is an excellent substrate for subtilisin itself. Activated esters, on the other hand, are substrates. 

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