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

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

Volume: 22 Issue: 6

Author(s): Anders S. R. Odum, Kjeld Olesen, Soren Ostergaard, Lars Thim, Inga Norby and Morten Meldal

Affiliation:

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

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 k_cat/K_M 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.

Cite this article as:

S. R. Odum Anders, Olesen Kjeld, Ostergaard Soren, Thim Lars, Norby Inga and Meldal Morten, Substrate Specificity Profiling of Peptidyl-Lys Metallopeptidase of Armillaria mellea by FRET Based Peptide Library, Protein & Peptide Letters 2015; 22 (6) . https://dx.doi.org/10.2174/0929866522666150407122152

DOI https://dx.doi.org/10.2174/0929866522666150407122152	Print ISSN 0929-8665
Publisher Name Bentham Science Publisher	Online ISSN 1875-5305