Thomas P. Graycar

Probing the specificity of the S1 binding site of M222 mutants of subtilisin B. lentus with boronic acid inhibitors

Abstract Specificity differences between the S1-pockets of subtilisin B. lentus (SBL), and its M222C/S mutants have been explored with boronic acid inhibitors. Similar binding trends were noted, with 2,4-dichlorophenylboronic acid being the best overall inhibitor for each enzyme. In addition, a correlation between inhibitor binding and the electrophilicity of boron was found for both the M222C and M222S enzymes. Specificity differences between the S 1-pockets of subtilisin from B. lentus (SBL), and its M222C/S mutants, have been explored with boronic acid inhibitors. Similar binding trends were noted, with 2,4-dichlorophenyl boronic acid being the best overall inhibitor for each enzyme. In addition, a correlation between inhibitor binding and the electrophilicity of boron was found for both M222C and M222S enzymes.

Altering the specificity of subtilisin B. Lentus by combining site-directed mutagenesis and chemical modification

The thiol side chain of the M222C mutant of the subtilisin from Bacillus lentus (SBL) has been chemically modified by methyl-, aminoethyl-, and sulfonatoethylthiosulfonate reagents. Introduction of charged residues into the active site of the enzyme reduced the catalytic efficiency with Suc-AAPF-pNA as the substrate, but resulted in better binding of sterically demanding boronic acid inhibitors.

Altering the proteolytic activity of subtilisin through protein engineering.

The utility of protein engineering in redesigning the structure of a protein to tailor its functional properties has been firmly established. In particular, the Bacillus serine protease subtilisin has proven to be a useful model protein for examining the use of systematic structural modification to incorporate novel functional properties into an enzyme.1.2 The list of properties that have been altered in subtilisin via such modification includes oxidative ~tability,”~ thermal ~tability,~ alkaline pH stability,h stability in organic ~olvent ,~ substrate specificity in aqueous nucleophile specificity,l2.l3 and pH activity profile.14 In addition to demonstrating the versatility of protein engineering, these studies have also provided valuable insight into the expected consequences of protein structure modification. For example, it is now recognized that while amino acid substitutions generally lead to only slight structural perturbations, these minor changes in structure can cause significant changes in protein function. Furthermore, it is apparent from several studies with subtilisin that multiple amino acid substitutions may additively affect a particular functional property. Provided with this extensive data base of structure-function relationships in subtilisin, thc cngineering of subtilisin for altered proteolytic activity is now being attempted. Increasing the proteolytic activity of subtilisin could boost the enzyme’s effectiveness as an additive to household laundry detergents. Subtilisin sold for use in laundry detergents accounts for the largest share of the worldwide industrial enzyme market with sales estimated for 1991 at

Protein Engineering of Subtilisin

200 million. Furthermore, the utility of subtilisin for peptide synthesis in aqueous systems can be enhanced by decreasing the enzyme’s proteolytic activity. This would alleviate the problem of low synthesis yields obtained due to proteolysis of the peptide product.

Structural Changes Leading to Increased Enzymatic Activity in an Engineered Variant of Bacillus Lentus Subtilisin

The use of enzymes as catalysts in organic synthesis reactions has expanded rapidly in recent years.1–5 In contrast to conventional organic reactions enzyme-catalyzed reactions offer the potential of highly stereo-selective or regioselective transformations. The value of enzymes in organic synthesis can be further attributed to the high reactivity and mild conditions characteristic of enzyme-catalyzed reactions. The increased availability and lower cost of enzymes resulting from the development of recombinant DNA technology has also stimulated interest in the use of enzymes for the production of fine organic chemicals. An apparent impediment to the widespread utilization of enzymes as commercial biocatalysts, however, is the difficulty encountered in optimizing their use in industrial processes. Although enzymes are capable of reacting with compounds having structures similar to that of their natural substrates, the catalytic efficiency of reactions involving nonnatural substrates is often suboptimal. In addition, the inherent instability of proteins and the sensitivity of enzyme activity to alterations in pH further limit their utility in industry. Hence, despite the known advantages of enzymes their use as biocatalysts is currently limited to less than 5% of the total industrial enzyme market.6 In the past, the only methods available to optimize the performance of an enzyme involved chemical modification of residues on the surface of the enzyme or else random mutagenesis of the corresponding gene. Now, however, the recent development of protein engineering has made it possible to redesign the structure of an enzyme and tailor its functional properties for a particular application, thereby greatly enhancing the potential to create novel industrial biocatalysts.

Engineering Altered Substrate Specificity into Subtilisin BPN

Much of the recent effort of subtilisin protein engineering has centered on the subtilisin from Bacillus lentus. This enzyme has higher alkaline performance than either subtilisin BPN’ from Bacillus amyloliquefaciens or subtilisin Carlsberg from Bacillus licheniformis. While the amino acid sequence of B. lentus subtilisin differs at 106 positions from subtilisin BPN’, including six deleted residues at positions 37a, 58, and 161 to 164, the three-dimensional structures of these subtilisins are very similar and it is possible to draw direct correlations between them.

Variants of bacillus sp. TS-23 alpha-amylase with altered properties

The discovery of restriction enzymes has allowed researchers to undertake far more exact analysis of DNA fragments. It has provided a means of extracting and manipulating specific segments of DNA. Segments of DNA coding for a particular protein can now be incorporated into the genome of a prokaryotic or eukaryotic host in a manner that provides for overexpression of that protein.