Subtilisin site-specific mutations

Subtilisin, 170 active site of, 171,173 autocorrelation function of, 216, 216 potential surfaces for, 218 site-specific mutations, 184, 185, 187-188 Sugars, see Oligosaccharides Surface-constrained solvent model, 125... 

Narhi et al. (1991) recently reported an enhancement in the thermal stability of aprA-subtilisin by three point mutations. The mutations were ASNi. SER and ASN. SER to prevent cyclisation with the adjacent glycines and ASN . ASP in the Ca binding loop. The mutant form also exhibits improved stability to detergent denaturation with little dependence on calcium concentration. Subtilisin 8350 (derived from subtilisin BPN via six site-specific mutations) was found to be 100 times more stable than the wild type enzyme in aqueous solution and 50 times more stable than the wild type in anhydrous dimethylformamide (Wong et al, 1990)... 

N. W. Whitlow, M. Bryan, P.N. Enzymes in organic synthesis—use of subtilisin and a highly stable mutant derived from multiple site-specific mutations. J. Am. Chem. Soc. 1990, 112 (3), 945-953. 

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. 

The construction of enzymes with new substrate specificities is now a realistic goal, and some novel approaches have been presented. For example, removal of an active-site histidine by the His-64 Ala mutation in subtilisin results in an enzyme with markedly reduced activity, but one which can be enhanced 400-fold with substrates containing histidine at the PI site (759). Apparently, the substrate histidine assists catalysis by partially compensating for the role of the lost active-site His-64. In a similar study, mutation of Lys-258 to Ala in aspartate aminotransferase produces an enzyme whose activity can be restored by exogenous amines (140). 

---