INACTIVATION BY OXYGEN AND STABILIZATION BY FLUOROCITRATE OF ACONITASE FROM ESCHERICHIA COLI

Abstract

More than 50% of the total aconitase activity in Escherichia coli was lost when cell-free extracts were prepared under air by a French press, by osmotic rupture of spheroplasts or by sonic treatment, as shown by the fact that the enzyme activity of the extracts prepared by sonic treatment under N₂ was more than twice that of the extracts prepared by the other methods. When the extracts prepared by sonic treatment under N₂ were gently shaken under air at 25°, about 70 to 80% of the original activity was lost over 30min, but a constant level of the enzyme activity remained even after prolonged incubation. The remaining aconitase activity after a long aerobic incubation was the same whether the extracts were prepared by sonic treatment under air or N₂. Aconitase was stable when cell-free extracts were incubated under anaerobic conditions or when fluorocitrate, in concentrations less than 1μM, was added during aerobic incubation. Citrate was less effective than fluorocitrate in preventing enzyme inactivation. Aconitase in the extracts, which had been incubated under air with fluorocitrate, was stable against oxygen inactivation and showed different V_max and Km values from those of the aconitase in the extracts that had been incubated under air without the addition of flourocitrate.