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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.


Inhibition of the Edmanase enzyme by a transition state analogue. (A) A series of fluorescence accumulation activity assays for the designed Edmanase enzyme in the presence of increasing inhibitor concentration are shown. PITC-derivatized Ala-AMC small molecules were used as the substrate for all assays. The enzyme is inhibited in a dose-dependent manner. (B). An inhibition curve fit to the data in panel A shows saturable inhibition with an IC50 of 1.1 mM. A schematic of the structure of the inhibitor is shown in the inset.
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fig07: Inhibition of the Edmanase enzyme by a transition state analogue. (A) A series of fluorescence accumulation activity assays for the designed Edmanase enzyme in the presence of increasing inhibitor concentration are shown. PITC-derivatized Ala-AMC small molecules were used as the substrate for all assays. The enzyme is inhibited in a dose-dependent manner. (B). An inhibition curve fit to the data in panel A shows saturable inhibition with an IC50 of 1.1 mM. A schematic of the structure of the inhibitor is shown in the inset.

Mentions: The two mutations described above provide evidence that the mechanism of catalysis for Edmanase is the substrate-assisted mechanism that was intended. To provide further support, we attempted to identify an inhibitor for Edmanase. Searching through commercially available compounds yielded three potential molecules with elaborated thiazole rings that structurally mimick our product analog. We characterized the activity of Edmanase upon PTC-Ala-AMC with increasing concentrations of inhibitors. One of the three candidates, 1-(2-anilino-5-methyl-1,3-thiazol-4-yl)-ethanone showed inhibition of Edmanase. The accumulation of cleaved substrate as a function of time for different concentrations of inhibitor are shown in Figure 7(A), and the inhibitory curve in Figure 7(B). The IC50 is high (1.14 mM), indicating that 1-(2-anilino-5-methyl-1,3-thiazol-4-yl)-ethanone is only a weak inhibitor. Nevertheless, the fact that a structural mimetic for our putative product analog inhibits the enzyme provides further evidence that we have realized our desired substrate-assisted mechanism.


Computer-aided design of a catalyst for Edman degradation utilizing substrate-assisted catalysis
Inhibition of the Edmanase enzyme by a transition state analogue. (A) A series of fluorescence accumulation activity assays for the designed Edmanase enzyme in the presence of increasing inhibitor concentration are shown. PITC-derivatized Ala-AMC small molecules were used as the substrate for all assays. The enzyme is inhibited in a dose-dependent manner. (B). An inhibition curve fit to the data in panel A shows saturable inhibition with an IC50 of 1.1 mM. A schematic of the structure of the inhibitor is shown in the inset.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig07: Inhibition of the Edmanase enzyme by a transition state analogue. (A) A series of fluorescence accumulation activity assays for the designed Edmanase enzyme in the presence of increasing inhibitor concentration are shown. PITC-derivatized Ala-AMC small molecules were used as the substrate for all assays. The enzyme is inhibited in a dose-dependent manner. (B). An inhibition curve fit to the data in panel A shows saturable inhibition with an IC50 of 1.1 mM. A schematic of the structure of the inhibitor is shown in the inset.
Mentions: The two mutations described above provide evidence that the mechanism of catalysis for Edmanase is the substrate-assisted mechanism that was intended. To provide further support, we attempted to identify an inhibitor for Edmanase. Searching through commercially available compounds yielded three potential molecules with elaborated thiazole rings that structurally mimick our product analog. We characterized the activity of Edmanase upon PTC-Ala-AMC with increasing concentrations of inhibitors. One of the three candidates, 1-(2-anilino-5-methyl-1,3-thiazol-4-yl)-ethanone showed inhibition of Edmanase. The accumulation of cleaved substrate as a function of time for different concentrations of inhibitor are shown in Figure 7(A), and the inhibitory curve in Figure 7(B). The IC50 is high (1.14 mM), indicating that 1-(2-anilino-5-methyl-1,3-thiazol-4-yl)-ethanone is only a weak inhibitor. Nevertheless, the fact that a structural mimetic for our putative product analog inhibits the enzyme provides further evidence that we have realized our desired substrate-assisted mechanism.

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.