Beneito-Cambra Miriam, Gareil Pierre, Badet Bernard, Badet-Denisot Marie-Ange, Delaunay Nathalie
Laboratory of Analytical and Bioanalytical Sciences and Miniaturization, UMR CBI 8231, ESPCI Paris, PSL Research University, 10 rue Vauquelin, 75005 Paris, France.
Chimie ParisTech, Laboratory of Physicochemistry of Electrolytes, Colloids and Analytical Sciences (PECSA), 75005 Paris, France.
J Chromatogr B Analyt Technol Biomed Life Sci. 2018 Jan 1;1072:130-135. doi: 10.1016/j.jchromb.2017.11.015. Epub 2017 Nov 12.
The enzyme glucosamine-6-phosphate synthase (GlmS) is an important point of metabolic control in biosynthesis of amino sugar-containing macromolecules and is therefore a potential target in order to design antibacterial and antifungal drugs. It has two oligomerization states, which are the active dimer and the inactive hexamer. For the first time, the potential of CE to separate and quantify the two forms was studied. After incubating GlmS with the d-glucosamine 6-phosphate (GlcN6P) inhibitor, an electrolyte based on sodium phosphate at pH 7.2 and an ionic strength of 100mM plus GlcN6P (either 2 or 20mM) allowed the hexamer-dimer separation. However, the displacement of the dimer/hexamer equilibrium during the analysis time prevented any improvement of the resolution when varying the effective separation length or the temperature of the analysis. Therefore, the use of a short-end CE method allowed the decrease in the analysis time to about 1min. Some parameters such as the temperature and the time of incubation and the ratio of the inhibitor and enzyme concentrations were studied. Then, it was also possible to test, very rapidly and with a very small amount, some molecules having an inhibition potential for the GlmS enzyme (arabinose-5-phosphate oxime, 2-amino-2-deoxy-d-glucitol 6-phosphate, and glucose-6-phosphate).
氨基葡萄糖-6-磷酸合酶（GlmS）是含氨基糖大分子生物合成中代谢控制的一个重要节点，因此是设计抗菌和抗真菌药物的一个潜在靶点。它有两种寡聚化状态，即活性二聚体和非活性六聚体。首次研究了毛细管电泳（CE）分离和定量这两种形式的潜力。用d-氨基葡萄糖6-磷酸（GlcN6P）抑制剂孵育GlmS后，在pH 7.2、离子强度为100mM的磷酸钠电解质加上GlcN6P（2或20mM）的条件下，可以实现六聚体-二聚体的分离。然而，在分析过程中二聚体/六聚体平衡的移动使得在改变有效分离长度或分析温度时分辨率无法得到任何提高。因此，采用短端CE方法可将分析时间缩短至约1分钟。研究了一些参数，如温度、孵育时间以及抑制剂与酶浓度的比例。然后，还能够非常快速且用量极少地测试一些对GlmS酶具有抑制潜力的分子（阿拉伯糖-5-磷酸肟、2-氨基-2-脱氧-d-葡萄糖醇6-磷酸和葡萄糖-6-磷酸）。
