Purification of a stable form of human aromatase in the absence of substrate: Effect of neuronal physiological pH changes on its substrate binding and activity

Recombinant human aromatase has been expressed in E. coli and for the first time purified in a stable form in the absence of any substrate or inhibitor: the active purified protein shows a Soret peak at 418 nm that completely shifts to 450 nm in the reduced CO-bound form. The protein retained the same activity of the one purified in the presence of the substrate androstenedione. The ability of the recombinant protein to bind the substrate androstenedione in the pH range 5.5-10 was investigated by UV-vis spectroscopy. The typical low-to-high spin transition, reflecting the displacement of the water ligand by substrate, was measured and a pH-dependency in substrate binding was found. Furthermore, a change of pH within 6.5 and 7.4, observed in physiological conditions in some active neurons, resulted in an increase of the KD and KM by 3 and 1.5 folds, respectively. The data presented suggest that aromatase activity can also be modulated in vivo by subtle changes of pH in neurons.