Substrate Specificity of Natural Variants and Genetically Engineered Intermediates of Bacillus Lentus Alkaline Proteases

Maurer, Karl‐Heinz; Markgraf, Martina; Goddette, Dean W.

doi:10.1007/978-1-4613-0319-0_26

Advances in Experimental Medicine and Biology

1996

DOI: 10.1007/978-1-4613-0319-0_26

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Substrate Specificity of Natural Variants and Genetically Engineered Intermediates of Bacillus Lentus Alkaline Proteases

Karl‐Heinz Maurer¹,

Martina Markgraf

Dean W. Goddette

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Cited by 4 publications

(2 citation statements)

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“…Furthermore, SBL's high-resolution crystal structure has been solved (30), it has been cloned, overexpressed, and purified (31), and its kinetic behavior has been well characterized (32). Also, studies toward altering its specificity (33)(34)(35) have been reported. Importantly, WT-SBL contains no natural cysteine residues and methanethiosulfonate reagents, therefore, react only with the introduced cysteine residue.…”

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Site-Directed Mutagenesis Combined with Chemical Modification as a Strategy for Altering the Specificity of the S₁ and S₁‘ Pockets of Subtilisin Bacillus lentus

et al. 1998

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By combining site-directed mutagenesis with chemical modification, we have altered the S1 and S1' pocket specificity of subtilisin Bacillus lentus (SBL) through the incorporation of unnatural amino acid moieties, in the following manner: WT --> Cysmutant + H3CSO2SR --> Cys-SR, where R may be infinitely variable. A paradigm between extent of activity changes and surface exposure of the modified residue has emerged. Modification of M222C, a buried residue in the S1' pocket of SBL, caused dramatic changes in kcat/KM, of an up to 122-fold decrease, while modification of S166C, which is located at the bottom of the S1 pocket and is partially surface exposed, effected more modest activity changes. Introduction of a positive charge at S166C does not alter kcat/KM, whereas the introduction of a negative charge results in lowered activity, possibly due to electrostatic interference with oxyanion stabilization. Activity is virtually unaltered upon modification of S156C, which is located toward the bottom of the S1 pocket and surface exposed and whose side chain is solvated. An unexpected structure-activity relationship was revealed for S166C-SR enzymes in that the pattern of activity changes observed with increasing steric size of R was not monotonic. Molecular modeling analysis was used to analyze this unprecedented structure-activity relationship and revealed that the position of the beta-carbon of Cys166 modulates binding of the P1 residue of the AAPF product inhibitor.

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Site-Directed Mutagenesis Combined with Chemical Modification as a Strategy for Altering the Specificity of the S₁ and S₁‘ Pockets of Subtilisin Bacillus lentus

et al. 1998

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“…The subtilisin from Bacillus lentus (SBL, EC 3.4.21.14) is well suited as an exploratory vehicle for evaluating the potential of this combined site-directed mutagenesis chemical modification approach since it is a well-characterized enzyme and is of synthetic ( , ) as well as industrial () interest. Furthermore, SBL's high-resolution crystal structure has been solved ( , ), and it has been cloned, overexpressed, and purified (), and its kinetic behavior well characterized ( − ). In addition, and importantly, wild-type (WT) SBL contains no natural cysteine residues, and methanethiosulfonate reagents therefore react only with the introduced cysteine residue.…”

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confidence: 99%

Toward Tailoring the Specificity of the S₁ Pocket of Subtilisin B. lentus: Chemical Modification of Mutant Enzymes as a Strategy for Removing Specificity Limitations

1999

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In both protein chemistry studies and organic synthesis applications, it is desirable to have available a toolbox of inexpensive proteases with high selectivity and diverse substrate preferences. Toward this goal, we have generated a series of chemically modified mutant enzymes (CMMs) of subtilisin B. lentus (SBL) possessing expanded S(1) pocket specificity. Wild-type SBL shows a marked preference for substrates with large hydrophobic P(1) residues, such as the large Phe P(1) residue of the standard suc-AAPF-pNA substrate. To confer more universal P(1) specificity on S(1), a strategy of chemical modification in combination with site-directed mutagenesis was applied. For example, WT-SBL does not readily accept small uncharged P(1) residues such as the -CH(3) side chain of alanine. Accordingly, with a view to creating a S(1) pocket that would be of reduced volume providing a better fit for small P(1) side chains, a large cyclohexyl group was introduced by the CMM approach at position S166C with the aim of partially filling up the S(1) pocket. The S166C-S-CH(2)-c-C(6)H(11) CMM thus created showed a 2-fold improvement in k(cat)/K(M) with the suc-AAPA-pNA substrate and a 51-fold improvement in suc-AAPA-pNA/suc-AAPF-pNA selectivity relative to WT-SBL. Furthermore, WT-SBL does not readily accept positively or negatively charged P(1) residues. Therefore, to improve SBL's specificity toward positively and negatively charged P(1) residues, we applied the CMM methodology to introduce complementary negatively and positively charged groups, respectively, at position S166C in S(1). A series of mono-, di-, and trinegatively charged CMMs were generated and all showed improved k(cat)/K(M)s with the positively charged P(1) residue containing substrate, suc-AAPR-pNA. Furthermore, virtually arithmetic improvements in k(cat)/K(M) were exhibited with increasing number of negative charges on the S166C-R side chain. These increases culminated in a 9-fold improvement in k(cat)/K(M) for the suc-AAPR-pNA substrate and a 61-fold improvement in suc-AAPR-pNA/suc-AAPF-pNA selectivity compared to WT-SBL for the trinegatively charged S166C-S-CH(2)CH(2)C(COO(-))(3) CMM. Conversely, the positively charged S166C-S-CH(2)CH(2)NH(3)(+) CMM generated showed a 19-fold improvement in k(cat)/K(M) for the suc-AAPE-pNA substrate and a 54-fold improvement in suc-AAPE-pNA/suc-AAPF-pNA selectivity relative to WT-SBL.

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Modulation of Infectivity in Phage Display as a Tool to Determine the Substrate Specificity of Proteases

et al. 2006

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Proteases play an important role in human and animal diseases. Rapid determination of substrate specificity is possible through the use of substrate phage display; however, current methods possess several drawbacks. They require phage-immobilization and cannot be used for infectivity-destroying or affinity tag-destroying proteases; this can make entire libraries useless. To overcome these limitations, here we introduce infectivity-modulated phage display (IMOP). IMOP uses a protease-resistant and infectivity-reducing tag fused to substrate-displaying polyvalent phages, and the specific cleavage of the substrate increases the infectivity of the phages by releasing the infectivity-reducing tag. The resulting phages were first tested with the infectivity-destroying detergent protease subtilisin; this resulted in a highly specific substrate at a 200-fold enrichment. In a second example, the protease ompT was used and led to an enrichment of the known double-arginine motif. The IMOP system thus substantially improves and simplifies previous systems.

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Substrate Specificity of Natural Variants and Genetically Engineered Intermediates of Bacillus Lentus Alkaline Proteases

Cited by 4 publications

References 19 publications

Site-Directed Mutagenesis Combined with Chemical Modification as a Strategy for Altering the Specificity of the S₁ and S₁‘ Pockets of Subtilisin Bacillus lentus

Site-Directed Mutagenesis Combined with Chemical Modification as a Strategy for Altering the Specificity of the S₁ and S₁‘ Pockets of Subtilisin Bacillus lentus

Toward Tailoring the Specificity of the S₁ Pocket of Subtilisin B. lentus: Chemical Modification of Mutant Enzymes as a Strategy for Removing Specificity Limitations

Modulation of Infectivity in Phage Display as a Tool to Determine the Substrate Specificity of Proteases

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Substrate Specificity of Natural Variants and Genetically Engineered Intermediates of Bacillus Lentus Alkaline Proteases

Cited by 4 publications

References 19 publications

Site-Directed Mutagenesis Combined with Chemical Modification as a Strategy for Altering the Specificity of the S1 and S1‘ Pockets of Subtilisin Bacillus lentus

Site-Directed Mutagenesis Combined with Chemical Modification as a Strategy for Altering the Specificity of the S1 and S1‘ Pockets of Subtilisin Bacillus lentus

Toward Tailoring the Specificity of the S1 Pocket of Subtilisin B. lentus: Chemical Modification of Mutant Enzymes as a Strategy for Removing Specificity Limitations

Modulation of Infectivity in Phage Display as a Tool to Determine the Substrate Specificity of Proteases

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Product

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Site-Directed Mutagenesis Combined with Chemical Modification as a Strategy for Altering the Specificity of the S₁ and S₁‘ Pockets of Subtilisin Bacillus lentus

Site-Directed Mutagenesis Combined with Chemical Modification as a Strategy for Altering the Specificity of the S₁ and S₁‘ Pockets of Subtilisin Bacillus lentus

Toward Tailoring the Specificity of the S₁ Pocket of Subtilisin B. lentus: Chemical Modification of Mutant Enzymes as a Strategy for Removing Specificity Limitations