title: Substrate Specificity Profiling of Peptidyl-Lys Metallopeptidase of Armillaria mellea by FRET Based Peptide Library.
abstract: Determining the substrate specificity of a protease is essential for developing assays, inhibitors and understanding the mechanisms of the enzyme. In this work, we have profiled the specificity of Peptidyl-Lys metallopeptidase, (LysN), of Armillaria mellea, by a synthetic fluorescence resonance energy transfer (FRET) positional-scanning library. The library was based on a reference sequence K(Abz)-S-A-Q-K-M-V-S-K(Dnp), where the fluorescent donor is 2-aminobenzamide and the quencher is N-2,4-dinitrophenyl. Each position was varied between 19 different amino acids one by one, to reveal the specificity of the protease. LysN exhibits strict specificity for lysine in S1', and has less specificity moving further away from the scissile bond. Additivity between the subsites was observed and the best substrate identified was K(Abz)-M-R-F-K-R-R-R-K(Dnp) with a kcat/KM of 42.6 µM/s. Based on a homology structure model the reference substrate was fitted into the active site using molecular dynamics to propose peptide-enzyme interactions.
authors.forename: Anders S R
authors.initials: AS
authors.lastname: Ødum
authors.forename: Kjeld
authors.initials: K
authors.lastname: Olesen
authors.forename: Søren
authors.initials: S
authors.lastname: Østergaard
authors.forename: Lars
authors.initials: L
authors.lastname: Thim
authors.forename: Inga
authors.initials: I
authors.lastname: Nørby
authors.affiliation: Global Research, Novo Nordisk AS, Novo Nordisk Park, 2760 Måløv, Denmark. kjdo@novonordisk.com.
authors.forename: Morten
authors.initials: M
authors.lastname: Meldal
mesh_terms: D000595:Amino Acid Sequence; D055392:Armillaria / Q000201:enzymology*; D004795:Enzyme Stability; D031541:Fluorescence Resonance Energy Transfer; D005656:Fungal Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D055672:Static Electricity; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase