Department of Chemistry, University of Konstanz, Universitätstraße 10, 78464, Konstanz, Germany.
Department of Pharmacy and Biotechnology, University of Bologna, Viale Risorgimento 4, 40136, Bologna, Italy; CSGI, University of Florence, Via della Lastruccia 3, 50019, Sesto Fiorentino, Firenze, Italy.
Biochimie. 2023 Oct;213:66-81. doi: 10.1016/j.biochi.2023.04.014. Epub 2023 May 17.
The Mycobacterium tuberculosis low-molecular weight protein tyrosine phosphatase (MptpA) is responsible for the inhibition of phagosome-lysosome fusion and is essential for the bacterium pathogenicity. This inhibition implies that M. tuberculosis is not exposed to a strongly acidic environment in vivo, enabling successful propagation in host cells. Remarkably, MptpA has been previously structurally and functionally investigated, with special emphasis devoted to the enzyme properties at pH 8.0. Considering that the virulence of M. tuberculosis is strictly dependent on the avoidance of acidic conditions in vivo, we analysed the pH-dependence of the structural and catalytic properties of MptpA. Here we show that this enzyme undergoes pronounced conformational rearrangements when exposed to acidic pH conditions, inducing a severe decrease of the enzymatic catalytic efficiency at the expense of phosphotyrosine (pTyr). In particular, a mild decrease of pH from 6.5 to 6.0 triggers a significant increase of K of MptpA for phosphotyrosine, the phosphate group of which we determined to feature a pKa equal to 5.7. Surface plasmon resonance experiments confirmed that MptpA binds poorly to pTyr at pH values < 6.5. Notably, the effectiveness of the MptpA competitive inhibitor L335-M34 at pH 6 does largely outperform the inhibition exerted at neutral or alkaline pH values. Overall, our observations indicate a pronounced sensitivity of MptpA to acidic pH conditions, and suggest the search for competitive inhibitors bearing a negatively charged group featuring pKa values lower than that of the substrate phosphate group.
结核分枝杆菌低分子量蛋白酪氨酸磷酸酶（MptpA）可抑制吞噬体-溶酶体融合，对细菌的致病性至关重要。这种抑制作用意味着结核分枝杆菌在体内不会暴露于强酸性环境中，从而能够在宿主细胞中成功繁殖。值得注意的是，MptpA 已经在结构和功能上进行了先前的研究，特别强调了在 pH8.0 时酶的特性。考虑到结核分枝杆菌的毒力严格依赖于在体内避免酸性条件，我们分析了 MptpA 的结构和催化特性对 pH 的依赖性。在这里，我们表明，当暴露于酸性 pH 条件下时，这种酶会发生明显的构象重排，导致酶催化效率严重降低，而磷酸酪氨酸（pTyr）的消耗增加。特别是，从 pH6.5 到 6.0 的轻微 pH 降低会导致 MptpA 对磷酸酪氨酸的 K 值显著增加，我们确定其磷酸基团的 pKa 值为 5.7。表面等离子体共振实验证实，MptpA 在 pH 值 < 6.5 时与 pTyr 的结合能力较差。值得注意的是，MptpA 的竞争性抑制剂 L335-M34 在 pH6 时的效果大大超过了在中性或碱性 pH 值下的抑制作用。总体而言，我们的观察结果表明 MptpA 对酸性 pH 条件非常敏感，并表明需要寻找具有比底物磷酸基团的 pKa 值更低的带负电荷基团的竞争性抑制剂。
