Serine proteases subtilisin

A practical enzymatic procedure using alcalase as biocatalyst has been developed for the synthesis of hydrophilic peptides.Alcalase is an industrial alkaline protease from Bacillus licheniformis produced by Novozymes that has been used as a detergent and for silk degumming. The major enzyme component of alcalase is the serine protease subtilisin Carlsberg, which is one of the fully characterized bacterial proteases. Alcalase has better stability and activity in polar organic solvents, such as alcohols, acetonitrile, dimethylformamide, etc., than other proteases. In addition, alcalase has wide specificity and both l- and o-amino acids that are accepted as nucleophiles at the p-1 subsite. Therefore, alcalase is a suitable biocatalyst to catalyse peptide bond formation in organic solvents under kinetic control without any racemization of the amino acids (Scheme 5.1). 

The application of CPO, HRP and CiP is limited to sterically unencumbered substrates and all these peroxidases produce the same absolute configuration of the chiral hydroperoxide. To overcome this limitation, the semisynthetic enzyme selenosubtilisin, a mimic for glutathione peroxidase, with the peptide framework of the serine protease subtilisin was developed by Bell and Hilvert. This semisynthetic peroxidase catalyzes the reduction of hydrogen peroxide and hydroperoxides in the presence of 5-mercapto-2-nitrobenzoic acid. It was utilized by Adam and coworkers and Schreier and coworkers for the kinetic resolution of racemic hydroperoxides (equation 17) . The results obtained were very promising. 

Khmelnitsky et al. were the first to observe the activating effects salt showed on enzymes in the nonaqueous environment [88]. As shown in Figure 3.7, the transesterification activity of the serine protease subtilisin Carlsberg in anhydrous solvents is strongly dependent on the KC1 content in a lyophilized enzyme preparation and increases sharply as the salt content is increased. This increase in activity was determined to be a result primarily of an increase in kcat and not a decrease in Km, as shown in (Table 3.4). 

An early discovery by Frederick Richards that turned out to be useful was that the protein could be cleaved between residues 20 and 21 by the bacterial serine protease, subtilisin. The resulting two polypeptides were separated and purified. They were enzymatically inactive individually, but regained the activity of the native enzyme when they were recombined. This work shows that strong, nonco-valent interactions occur that can hold protein chains together even when one of the peptide links is cut. It also makes it possible to modify specific amino acid residues of the two polypeptide chains independently and to explore how each residue contributes to the reassembly of the protein and the recovery of enzymatic activity. 

Kano, H., Taguchi, S., and Momose, H. (1997). Cold adaptation of a mesophilic serine protease, subtilisin, by in vitro random mutagenesis. Appl. Microbiol. Biotechnol, 47, 46-51. 

In the catalytic mechanism of the serine protease subtilisin, the tetrahedral intermediate is believed to be stabilized by a hydrogen bond to the side chain of Asn 155. Replacement of Asn 155 with Gly left the substrate binding unaffected, but inhibited the catalytic step, confirming the proposed mechanism. 

An illustrative example of a change in chemoselectivity is the inversion of substrate specificity of the serine protease Subtilisin Carlsberg in the transesterification reaction of ethyl esters of A-acetyl-L-serine and A-acetyl-L-phenylalanine with 1-propanol, measured in twenty anhydrous organic solvents. The enzyme-catalysed reaction with the serine substrate is strongly favoured in dichloromethane, while the reaction with the phenylalanine substrate is preferred in t-butylamine, with a 68-fold change in substrate specificity [313]. 

Jackson, S. E., Fersht, A. R. (1993) Contribution of Long-Range Electrostatic Interactions to the Stabilization of the Catalytic Transition State of the Serine Protease Subtilisin BPN , Biochemistry 32, 13909-13916. 

Jackson SE, Fersht AR (1993) Contribution of long-range electrostatic interactions to the stabilization of the catalytic transition state of the serine protease subtilisin BPN . Biochemistry 32(50) 13909-13916... 

Directed evolution has also been very effective for increasing enzyme activity in organic solvents 14> For example, the serine protease subtilisin can catalyze specific peptide syntheses and transesterification reactions, but organic solvents are required to drive the reaction towards synthesis. Sequential rounds of error-prone PCR and visual screening yielded a subtilisin variant with twelve amino acid substitutions that was 471 times more active than wild-type in 60% dimethylforma-mide (DMF)[145- 22° this enzyme is much more effective for peptide and polymer synthesis. 

Enzymes are highly specific catalysts. The nature of this specificity is believed to result from structural and electrostatic complementarity between the enzyme and its substrate. The serine protease, subtilisin, is being extensively studied as a model system to explore the effects of single amino acid substitutions on its structure and function Q). The gene for Bacillus amyloliquefaciens subtilisin has been expressed and secreted in B. subtilis 12).A site-directed mutagenesis scheme, cassette mutagenesis ( ), has been used to produce a series of subtilisin variants that are more resistant to oxidants (4), and have altered stability ( ), specificity, and specific activity. 

B. subtilis produces at least eight Eprs at the end of the exponential phase of growth in liquid culture [80]. These have been identified as the alkaline serine protease subtilisin (AprE) [81,82], the neutral protease (NprE) [83], the minor Epr [84,85], the bacillopeptidase F (Bpr) [86], the Vpr protease [87], the metalloprotease (Mpr) [86], the NprB [83], and the cell wall-associated extracellular protease WprA [88] (Figure 7.2). Proteomics and transcriptomics studies have shown the involvement of the DegS-DegU two-component regulatory system in the expression of several of these exoproteases [89,90]. 

For example, Kraut and coworkers [43] have proposed a stereochemical mechanism for the action of the serine protease subtilisin. They use a considerable amount of structural information about the enzyme and several of its stable complexes nevertheless, the key element of the hypothesis is that the complex of the (very unstable) transition state of the substrate and the enzyme is stabilized by nonbonded interactions to a much greater extent than is any stable complex. This reliance on the unstable transition state [44] makes experimental verification of the mechanism extremely difficult. 

It has been suggested that the active sites in proteins are better conserved than the overall fold [27]. If so, then one should be able to identify not only distant ancestors with the same global fold and same biochemical activity, but also proteins with similar functions but different global folds. Nussinov and coworkers empirically demonstrated that the active sites of eukaryotic serine proteases, subtilisins, and sulfhydryl proteases exhibit similar structural motifs [216]. Furthermore, in a recent modeling study of S. cerevisiae proteins, active... 

Corresponding equations were derived for the cysteine proteases ficin, actinidin, bromelain B and D, and the serine proteases subtilisin, chymotrypsin, and trypsin. In all cases, the coefficient of the electronic a term is around 0.4-0.7. Whereas the other regression coefficients in equations (16) and (17) are also relatively similar, the constant terms of both equations differ by about 1.8 log units, which indicates that the lower lipophilicity of the mesyl group, as compared with a much more lipophilic benzoyl group, is responsible for the lower binding affinities of the mesyl amides. To prove this hypothesis, a series of (4-X-Phe)-CONHCH2COO-pyrid-3-yl analogs 12 was synthesized and tested. As expected, lipophilicity determines the structure-activity relationship in this part of the molecules (equation IS). " " ... 

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