Subtilisin primary structure

Subtilisins Primary Structure, Chemical and Physical Properties Francis S. Markland, Jr. and Emil L. Smith... 

An example of this kind of work involves the enzyme subtilisin, frequently used as an additive in laundry detergents because it attacks the proteins that soil clothing. The problem, however, is that subtilisin is easily destroyed by bleaches with which a detergent is often used. Research showed that subtilisin is sensitive to bleach because a single amino acid in its primary structure—a methionine at position 22—is destroyed by bleach. By replacing this methionine with an amino acid that is not sensitive to attack by bleach, then, researchers were able to synthesize a new form of subtilisin that did not degrade in the presence of bleach for use in laundry detergents. 

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.

Comparative studies on the primary structures of homologous P. from different species (e.g. hemoglobin from vertebrates, see Homologous proteins) or analogous P. (e. g. subtilisin from Bacillus subtilis and mammalian trypsin) have made a valuable biochemical contribution to questions of divergent and convergent evolution. However, for an explanation of P. function and behavior, especially the mechanism of enzyme action, the primary structure alone is insufficient, and a knowledge of secondary and tertiary structure is needed. 

The group is typically divided into two families the subtilisin-like and chymotrypsin-like groups (7). The chymoti sin-like group are represented by members in all organisms whereas the subtilisin-like members are found in only prokaryotes. Although both families have dissimilar primary and tertiaiy structures they share a common catalytic mechanism (8,9),... 

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. 

Subtilisin, a bacterial proteolytic enzyme originally isolated from Bacillt4S subtilus, is a serine protease. Even though its primary and tertiary structures bear no discernible relationship to chymotrypsin, the active site groups and the... 

Oxidation of Met residues has significantly affected biological activity in some instances (subtilisin, E. colt ribosomal protein L12), but has shown no effect in others (ribonuclease, Kunitz trypsin inhibitor). As is the case with degradation at Asx, the rate of oxidation at select residues within a protein is dependent upon higher-order structure, presumably because of solvent inaccessibility or steric hindrance. Howevei unlike the case of Asx degradation, there are no primary sequence or other structural motifs found to strongly correlate with oxidation of Met. Other amino add residues susceptible to oxidation are Cys, His, Trp, and Tyr. 

R. J. Kazlauskas, A.N.E. Weissfloch, A structure-based rationalization of the enantiopreference of subtilisin toward secondary alcohols and isosteric primary amines, 1. Mol. Catal. B Enzymatic 3 (1997) 65-72. 

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