Helmholtz Zentrum München, Institute of Structural Biology, Neuherberg, Germany.
Malopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland.
J Enzyme Inhib Med Chem. 2021 Dec;36(1):1267-1281. doi: 10.1080/14756366.2021.1937619.
Mirolysin is a secretory protease of , a member of the dysbiotic oral microbiota responsible for periodontitis. In this study, we show that mirolysin latency is achieved by a "cysteine-switch" mechanism exerted by Cys23 in the N-terminal profragment. Mutation of Cys23 shortened the time needed for activation of the zymogen from several days to 5 min. The mutation also decreased the thermal stability and autoproteolysis resistance of promirolysin. Mature mirolysin is a thermophilic enzyme and shows optimal activity at 65 °C. Through NMR-based fragment screening, we identified a small molecule (compound (cpd) ) that blocks promirolysin maturation and functions as a competitive inhibitor ( = 3.2 µM), binding to the S1' subsite of the substrate-binding pocket. Cpd shows superior specificity and does not interact with other proteases or Lys/Arg-specific proteases.
纤溶酶原激活物是一种分泌型蛋白酶，属于致牙周病的口腔失调微生物群的一员。在这项研究中，我们表明纤溶酶原激活物的潜伏期是通过 N 端前片段中的 Cys23 发挥的“半胱氨酸开关”机制实现的。Cys23 的突变将酶原激活所需的时间从数天缩短至 5 分钟。该突变还降低了前纤溶酶原的热稳定性和自身水解抗性。成熟的纤溶酶原是一种嗜热酶，在 65°C 时表现出最佳活性。通过基于 NMR 的片段筛选，我们鉴定出一种小分子（化合物 (cpd)），它可阻断前纤溶酶原的成熟并作为竞争性抑制剂发挥作用（ = 3.2µM），与底物结合口袋的 S1'亚位点结合。Cpd 表现出更高的特异性，并且不与其他蛋白酶或 Lys/Arg 特异性蛋白酶相互作用。
