title: Catalytic activation of pre-substrates via dynamic fragment assembly on protein templates.
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.
authors.affiliation: Institute of Pharmacy, Medicinal Chemistry, University of Leipzig, Brüderstraße 34, 04103 Leipzig, Germany.
authors.forename: Edyta
authors.initials: E
authors.lastname: Burda
authors.affiliation: 1] Institute of Pharmacy, Medicinal Chemistry, University of Leipzig, Brüderstraße 34, 04103 Leipzig, Germany [2] Institute of Pharmacy, Medicinal Chemistry, Freie Universität Berlin, Königin-Luise-Straße 2+4, 14195 Berlin, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Rademann
mesh_terms: D001665:Binding Sites; D055162:Biocatalysis; D020134:Catalytic Domain; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D057057:Serine Proteases / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D010446:Peptide Fragments; D057057:Serine Proteases