Catalytic activation of pre-substrates via dynamic fragment assembly on protein templates

Edyta Burda and Jörg Rademann ()
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Edyta Burda: Institute of Pharmacy, Medicinal Chemistry, University of Leipzig
Jörg Rademann: Institute of Pharmacy, Medicinal Chemistry, University of Leipzig

Nature Communications, 2014, vol. 5, issue 1, 1-9

Abstract: Abstract Sensitive detection of small molecule fragments binding to defined sites of biomacromolecules is still a considerable challenge. Here we demonstrate that protein-binding fragments are able to induce enzymatic reactions on the protein surface via dynamic fragment ligation. Fragments binding to the S1 pocket of serine proteases containing a nitrogen, oxygen or sulphur nucleophile are found to activate electrophilic pre-substrates through a reversible, covalent ligation reaction. The dynamic ligation reaction positions the pre-substrate molecule at the active site of the protein thereby inducing its enzymatic cleavage. Catalytic activation of pre-substrates is confirmed by fluorescence spectroscopy and by high-performance liquid chromatography. The approach is investigated with 3 pre-substrates and 14 protein-binding fragments and the specific activation and the templating effect exerted by the enzyme is quantified for each protease–fragment–pre-substrate combination. The described approach enables the site-specific identification of protein-binding fragments, the functional characterization of enzymatic sites and the quantitative analysis of protein template-assisted ligation reactions.

Date: 2014
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DOI: 10.1038/ncomms6170

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