Subtilisin site-directed mutagenesis

Bryan, P., et al. Site-directed mutagenesis and the role of the oxyanion hole in subtilisin. Proc. Natl. Acad. Sci. USA 83 3743-3745, 1986. 

The family of serine proteases has been subjected to intensive studies of site-directed mutagenesis. These experiments provide unique information about the contributions of individual amino acids to kcat and KM. Some of the clearest conclusions have emerged from studies in subtilisin (Ref. 9), where the oxyanion intermediate is stabilized by t>e main-chain hydrogen bond of Ser 221 and an hydrogen bond from Asn 155 (Ref. 2). Replacement of Asn 155 (e.g., the Asn 155— Ala 155 described in Fig. 7.9) allows for a quantitative assessment of the effect of the protein dipoles on Ag. ... 

Using X-ray structure data, amino acids which were believed to affect the desired characteristics were identified. Using either site-directed mutagenesis or "cassette" mutagenesis (8) different amino acid substitutions were made in the subtilisin structural gene (2,9). 

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. 

Besides these rather complex coenzyme-dependent enzymes, the none-coenzyme requiring protease subtilisin is the most extensively mutated enzyme. The substrate specificity of the enzyme as well as its dependence on pH and its stability were altered by site-directed mutagenesis [72-78]. As the knowledge about exact details of the structure and active site of the enzyme is essential for the application of this method, progress in this field is difficult to achieve. Site-directed mutagenesis as a means of catalyst improvements will be used only after extensive application of conventional optimization procedures. 

Scheme 5.1.6. Controlled site-selective modification of subtilisin by a combined site-directed mutagenesis chemical modification approach.

Since there are strict stereochemical requirements for the relative positions and orientations of the two participating cysteine residues,11 addition of new disulfides to existing proteins by site-directed mutagenesis has not always produced the desired increase in stability. Introduction of disulfide bonds has been attempted for phage T4 lysozyme,4-71 phage A repressor,81 dihydrofolate reductase,91 and subtilisins.10-131 Among them the most extensive study has been performed on T4 lysozyme, and enhancement of protein stability has been successful. 

Introduction of a Disulfide Bond into Subtilisin by Site-directed Mutagenesis... 

Narinx, E., Baise, E., and Gerday, C. (1997). Subtilisin from psychrophilic antarctic bacteria characterization and site-directed mutagenesis of residues possibly involved in the adaptation to cold. Protein Engineering, 10, 1271—1279. 

The techniques of molecular biology discussed in Chapter 6 have permitted detailed examination of the catalytic triad. In particular, site-directed mutagenesis has been used to test the contribution of individual amino acid residues to the catalytic power of an enzyme. Subtilisin has been extensively studied by this method. Each of the residues within the catalytic triad, consisting of aspartic acid 32, histidine 64, and serine 221, has been individually converted into alanine, and the ability of each mutant enzyme to cleave a model substrate has been examined (Figure 9.16). As expected, the conversion of active-site serine 221 into alanine dramatically reduced catalytic power the value of k fell to less than one-millionth of its value for the wild-type enzyme. The value of. Sf was essentially unchanged its increase by no more than a factor of two indicated that substrate binding is not significantly affected. The mutation of histidine 64 to alanine... 

Because the oxyanion hole of subtilisin includes a side-chain NH group in addition to backbone NH groups, it is possible to probe the importance of the oxyanion hole for catalysis by site-directed mutagenesis. The mutation of asparagine 155 to glycine reduced the value of k to 0.2% of its wild-type value but increased the value of. ST by only a factor of two. 

Figure 9.16. Site-Directed Mutagenesis of Subtilisin. Residues of the catalytic triad were mutated to alanine, and the activity of the mutated enzyme was measured. Mutations in any component of the catalytic triad cause a dramatic loss of enzyme activity. Note that the activity is displayed on a logarithmic scale. The mutations are identified as follows the first letter is the one-letter abbreviation for the amino acid being altered the number identifies the position of the residue in the primary structure and the second letter is the one-letter abbreviation for the amino acid replacing the original one.

Some examples of hydrolases are listed in Table 7.3 according to the industrial sector in which they are used. Most of these hydrolases operate in the hydrolytic mode, since only a few processes are currently known that make use of the synthetic power of hydrolases. Increasingly, however, these enzymes are being tailored by genetic tools such as random or site-directed mutagenesis and overexpression to lower the operational costs of the biocatalyst and to obtain enzymes of high purity, as well as to meet specific customer requirements such as stability, substrate specificity, and optimal pH and temperature of operation. A typical example is the protease subtilisin from Bacillus subtilus, which was made bleach-resistant by replacing one bleach-sensitive amino acid (cysteine) by the chemically inert alanine [12]. 

Martinez P, Van Dam ME, Robinson AC, Chen K, and Arnold EH. Stabilization of Subtilisin E in Organic Solvents by Site-directed Mutagenesis. Biotechnol Bioeng 1992 39 141-147. 

Combining site-directed mutagenesis strategies with chemical modification is a popular tool in both enzyme engineering and mechanistic studies. This has often been applied to the subtilisin from Bacillus lentus (SBL), or savinase. Subtilisins are... 

Subtilisins are a family of serine proteases, the most important members of which are subtilisin Carlsberg (from Bacillus licheniformis) and subtilisin BPN (from Bacillus amyloliquefaciens)luoK Both enzymes are alkaline proteases with a pH optimum of 6-9. Because of their industrial importance, both subtilisin Carlsberg and subtilisin BPP have been studied intensively and are produced on a large scale. The crystal structures of both subtilisins have been determined1821. Directed evolution and site-directed mutagenesis and chemical modification of subtilisin were carried out in order to influence the stability, activity and enantioselectivity of the enzyme, in particular in organic solvents11111. As in the case of other enzymes,... 

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