First investigations for the characterization of glucosamine-6-phosphate synthase by capillary electrophoresis
Abstract 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). Highlights Glucosamine-6-phosphate synthase exists as an active dimer or an inactive hexamer. For the first time, the potential of CE to separate the two forms was demonstrated. A short-end CE method led to an analysis time of about 1min. The effect of some incubation conditions and the inhibitor/enzyme ratio was studied. 3 potential enzyme inhibitors were tested very rapidly and with very small amounts.
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