Phosphoenolpyruvate carboxykinase ferroactivator 1. Mechanism of action and identity with glutathione peroxidase.

A cytosolic protein factor (ferroactivator) facilitates the activation of phosphoenolpyruvate carboxykinase by ferrous ions (Bentle, L. A., and Lardy, H. A. (1977) J. Biol. Chem. 252, 1431-1440). We have extended our studies on the interaction of Fe2+ with this enzyme to establish the conditions under which it is an activator or an inhibitor. Preincubation of phosphoenolpyruvate carboxykinase with Fe2+ and dithiothreitol resulted in irreversible loss of enzyme activity within minutes of Fe2+ addition. This was attributed to an active oxygen species produced by aerobic oxidation of the divalent metal ion in the presence of dithiothreitol as suggested by lack of inhibition in preincubation experiments with Fe2+ under mildly acidic pH; ferroactivation by many H2O2 scavenging enzymes; and lack of inhibition on preincubation under anaerobic conditions. We conclude that Fe2+ per se can activate phosphoenolpyruvate carboxykinase and that ferroactivator protein helps to overcome the deleterious effects of aerobic oxidation. Mechanistic details of ferroactivation and a comparison of the known properties of ferroactivator indicated the similarity of this protein with rat liver glutathione peroxidase. The identity of ferroactivator as glutathione peroxidase was confirmed by the demonstration of catalytic activity, selenium content, and immunological cross-reactivity.