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Computer-aided design of a catalyst for Edman degradation utilizing substrate-assisted catalysis

View Article: PubMed Central

ABSTRACT

Molecular biology has been revolutionized by the miniaturization and parallelization of DNA sequencing assays previously performed on bulk samples. Many of these technologies rely on biomolecular reagents to facilitate detection, synthesis, or labeling of samples. To aid in the construction of analogous experimental approaches for proteins and peptides, we have used computer-aided design to engineer an enzyme capable of catalyzing the cleavage step of the Edman degradation. We exploit the similarity between the sulfur nucleophile on the Edman reagent and the catalytic cysteine in a naturally occurring protease to adopt a substrate-assisted mechanism for achieving controlled, step-wise removal of N-terminal amino acids. The ability to expose amino acids iteratively at the N-terminus of peptides is a central requirement for protein sequencing techniques that utilize processive degradation of the peptide chain. While this can be easily accomplished using the chemical Edman degradation, achieving this activity enzymatically in aqueous solution removes the requirement for harsh acid catalysis, improving compatibility with low adsorption detection surfaces, such as those used in single molecule assays.

No MeSH data available.


Catalytic efficiency of Edmanase and attenuation mutants. The catalytic efficiencies (kcat/KM) of the negative controls (BSA) and point mutants are compared to that of Edmanase. The substrate in each case was Ala-AMC, either untreated (PITC-) or derivatized with PITC (PITC+). BSA indicates values measured when Edmanase was replaced with BSA. The point mutants G25V and H159A serve to verify the intended catalytic mechanism of Edmanase by excluding the nucleophilic sulfur from the active site, and by removing the putative general base of the catalytic triad, respectively.
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fig05: Catalytic efficiency of Edmanase and attenuation mutants. The catalytic efficiencies (kcat/KM) of the negative controls (BSA) and point mutants are compared to that of Edmanase. The substrate in each case was Ala-AMC, either untreated (PITC-) or derivatized with PITC (PITC+). BSA indicates values measured when Edmanase was replaced with BSA. The point mutants G25V and H159A serve to verify the intended catalytic mechanism of Edmanase by excluding the nucleophilic sulfur from the active site, and by removing the putative general base of the catalytic triad, respectively.

Mentions: We expressed and characterized the four-fold mutant using an assay that follows the accumulation of fluorescence as Edmanase cleaves the PTC-amino acid from the “N-terminus” of a PTC-Ala-AMC substrate (see Methods section). As negative controls, we also performed the assay in the presence of BSA instead of Edmanase and using substrate that had not been derivatized with PITC (denoted PITC- in Fig. 5). Low levels of fluorescence accumulation are observed with BSA (Fig. 5, BSA PITC- and BSA PITC+), and are attributed to the uncatalyzed rate of Ala-AMC bond cleavage under mild aqueous conditions. Cleavage of Ala-AMC (PITC-) substrate by Edmanase is comparable to that of the PITC+ substrate with BSA. There is a pronounced increase in catalytic efficiency for the PITC+ substrate compared to PITC- in the presence Edmanase of 282-fold (Fig. 5, Table I). Thus, robust cleavage of the Ala-AMC bond requires both derivatization of the small molecule with PITC and the presence of Edmanase.


Computer-aided design of a catalyst for Edman degradation utilizing substrate-assisted catalysis
Catalytic efficiency of Edmanase and attenuation mutants. The catalytic efficiencies (kcat/KM) of the negative controls (BSA) and point mutants are compared to that of Edmanase. The substrate in each case was Ala-AMC, either untreated (PITC-) or derivatized with PITC (PITC+). BSA indicates values measured when Edmanase was replaced with BSA. The point mutants G25V and H159A serve to verify the intended catalytic mechanism of Edmanase by excluding the nucleophilic sulfur from the active site, and by removing the putative general base of the catalytic triad, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4380987&req=5

fig05: Catalytic efficiency of Edmanase and attenuation mutants. The catalytic efficiencies (kcat/KM) of the negative controls (BSA) and point mutants are compared to that of Edmanase. The substrate in each case was Ala-AMC, either untreated (PITC-) or derivatized with PITC (PITC+). BSA indicates values measured when Edmanase was replaced with BSA. The point mutants G25V and H159A serve to verify the intended catalytic mechanism of Edmanase by excluding the nucleophilic sulfur from the active site, and by removing the putative general base of the catalytic triad, respectively.
Mentions: We expressed and characterized the four-fold mutant using an assay that follows the accumulation of fluorescence as Edmanase cleaves the PTC-amino acid from the “N-terminus” of a PTC-Ala-AMC substrate (see Methods section). As negative controls, we also performed the assay in the presence of BSA instead of Edmanase and using substrate that had not been derivatized with PITC (denoted PITC- in Fig. 5). Low levels of fluorescence accumulation are observed with BSA (Fig. 5, BSA PITC- and BSA PITC+), and are attributed to the uncatalyzed rate of Ala-AMC bond cleavage under mild aqueous conditions. Cleavage of Ala-AMC (PITC-) substrate by Edmanase is comparable to that of the PITC+ substrate with BSA. There is a pronounced increase in catalytic efficiency for the PITC+ substrate compared to PITC- in the presence Edmanase of 282-fold (Fig. 5, Table I). Thus, robust cleavage of the Ala-AMC bond requires both derivatization of the small molecule with PITC and the presence of Edmanase.

View Article: PubMed Central

ABSTRACT

Molecular biology has been revolutionized by the miniaturization and parallelization of DNA sequencing assays previously performed on bulk samples. Many of these technologies rely on biomolecular reagents to facilitate detection, synthesis, or labeling of samples. To aid in the construction of analogous experimental approaches for proteins and peptides, we have used computer-aided design to engineer an enzyme capable of catalyzing the cleavage step of the Edman degradation. We exploit the similarity between the sulfur nucleophile on the Edman reagent and the catalytic cysteine in a naturally occurring protease to adopt a substrate-assisted mechanism for achieving controlled, step-wise removal of N-terminal amino acids. The ability to expose amino acids iteratively at the N-terminus of peptides is a central requirement for protein sequencing techniques that utilize processive degradation of the peptide chain. While this can be easily accomplished using the chemical Edman degradation, achieving this activity enzymatically in aqueous solution removes the requirement for harsh acid catalysis, improving compatibility with low adsorption detection surfaces, such as those used in single molecule assays.

No MeSH data available.