Konstantinov Mihail, Kaluzhskiy Leonid, Yablokov Evgeniy, Zhdanov Dmitry, Ivanov Alexis, Toropygin Ilya
Institute of Biomedical Chemistry, 10 Building 8, Pogodinskaya Street, 119121 Moscow, Russia.
Int J Mol Sci. 2025 Sep 4;26(17):8603. doi: 10.3390/ijms26178603.
β-Lytic protease (Blp) and protease L5 are enzymes from bacteria with distinct proteolytic and bacteriolytic activities. To characterize their substrate specificity, we employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis following hydrolysis of fractionated protein mixtures. Heatmaps and sequence logos revealed a pronounced specificity of Blp towards glycine and lysine residues, while L5 preferentially cleaved non-polar residues such as methionine, phenylalanine, and leucine. Notably, proline was frequently observed at the P2 position in L5 substrates. Comparative analysis with trypsin revealed that L5 generated significantly shorter peptides, whereas Blp produced fragments similar in length to tryptic peptides. These findings indicate different cleavage preferences and suggest potential applications for these enzymes in proteomic analysis.
β-溶菌蛋白酶（Blp）和蛋白酶L5是来自细菌的具有不同蛋白水解和溶菌活性的酶。为了表征它们的底物特异性，我们在对分级分离的蛋白质混合物进行水解后采用了液相色谱-串联质谱（LC-MS/MS）分析。热图和序列标识显示Blp对甘氨酸和赖氨酸残基具有明显的特异性，而L5优先切割甲硫氨酸、苯丙氨酸和亮氨酸等非极性残基。值得注意的是，脯氨酸在L5底物的P2位置经常出现。与胰蛋白酶的比较分析表明，L5产生的肽明显更短，而Blp产生的片段长度与胰蛋白酶肽相似。这些发现表明了不同的切割偏好，并暗示了这些酶在蛋白质组分析中的潜在应用。
