Ødum Anders S R, Olesen Kjeld, Østergaard Søren, Thim Lars, Nørby Inga, Meldal Morten
Global Research, Novo Nordisk AS, Novo Nordisk Park, 2760 Måløv, Denmark.
Protein Pept Lett. 2015;22(6):514-24. doi: 10.2174/0929866522666150407122152.
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.
确定蛋白酶的底物特异性对于开发检测方法、抑制剂以及理解酶的作用机制至关重要。在这项工作中，我们通过合成荧光共振能量转移（FRET）位置扫描文库，分析了蜜环菌肽基赖氨酸金属肽酶（LysN）的特异性。该文库基于参考序列K(Abz)-S-A-Q-K-M-V-S-K(Dnp)，其中荧光供体是2-氨基苯甲酰胺，淬灭剂是N-2,4-二硝基苯基。每个位置逐一在19种不同氨基酸之间变化，以揭示蛋白酶的特异性。LysN对S1'位点的赖氨酸表现出严格的特异性，远离切割键时特异性降低。观察到亚位点之间的加和性，鉴定出的最佳底物是K(Abz)-M-R-F-K-R-R-R-K(Dnp)，其kcat/KM为42.6 μM/s。基于同源结构模型，使用分子动力学将参考底物拟合到活性位点，以提出肽-酶相互作用。
