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Substrate Specificity Profiling of Peptidyl-Lys Metallopeptidase of Armillaria mellea by FRET Based Peptide Library

[ Vol. 22 , Issue. 6 ]


Anders S. R. Odum, Kjeld Olesen, Soren Ostergaard, Lars Thim, Inga Norby and Morten Meldal   Pages 514 - 524 ( 11 )


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.


Enzyme kinetics, fluorescence resonance energy transfer (FRET), homology modeling, metalloprotease, proteolysis, substrate specificity.


Global Research, Novo Nordisk AS, Novo Nordisk Park, 2760 Måløv, Denmark.

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