School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, Norfolk NR47TJ, UK.
Anal Biochem. 2009 May 1;388(1):91-6. doi: 10.1016/j.ab.2009.02.015. Epub 2009 Feb 20.
Mycothiol (MSH) is the principal low-molecular-weight thiol, unique to mycobacteria and other actinomycetes, that performs a role analogous to glutathione found in other organisms. MSH plays a key role in oxidative stress management and is oxidized to the dimeric mycothiol disulfide (MSSM) in the process. NADPH-dependent mycothiol disulfide reductase (Mtr) helps to maintain an intracellular reducing environment by reducing MSSM back to MSH. Mtr inhibition studies are currently impaired by limited availability of MSSM. Substrate demands are particularly high in time-dependent inhibition assays. Here we report an assay that chemically recycles a mixed disulfide substrate analogue in situ, thereby greatly reducing the substrate quantities needed for such assays. This has enabled the development of a continuous assay where linear reaction rates can be maintained for 40 min or longer using minimal substrate concentrations (5 microM versus a substrate K(m) value of 268 microM). In this manner, substrate requirements are reduced by orders of magnitude. Characterization of a novel time-dependent inhibitor, 2-(5-bromo-2-methoxyphenyl)acrylonitrile, is also demonstrated using these procedures.
分枝菌酸（MSH）是一种主要的低分子量巯基化合物，仅存在于分枝杆菌和其他放线菌中，其功能类似于其他生物体中的谷胱甘肽。MSH 在氧化应激管理中发挥着关键作用，并在此过程中被氧化为二聚分枝菌酸二硫醚（MSSM）。NADPH 依赖性分枝菌酸二硫键还原酶（Mtr）通过将 MSSM 还原回 MSH 来帮助维持细胞内的还原环境。由于 MSSM 的有限可用性，分枝菌酸二硫键还原酶抑制研究目前受到阻碍。在时间依赖性抑制测定中，底物需求特别高。在这里，我们报告了一种在原位化学循环混合二硫键底物类似物的测定方法，从而大大减少了此类测定所需的底物数量。这使得可以开发一种连续测定方法，其中使用最小的底物浓度（5μM 对 268μM 的底物 Km 值）可以维持 40 分钟或更长时间的线性反应速率。通过这种方式，底物需求降低了几个数量级。还使用这些程序对新型时间依赖性抑制剂 2-（5-溴-2-甲氧基苯基）丙烯腈进行了表征。
