RESUMEN
Ribosomal function is dependent on multiple proteins. The ABCE1 ATPase, a unique ABC superfamily member that bears two Fe4S4 clusters, is crucial for ribosomal biogenesis and recycling. Here, the ATPase activity of the Pyrococcus abyssi ABCE1 (PabABCE1) was studied using both apo- (without reconstituted Fe-S clusters) and holo- (with full complement of Fe-S clusters reconstituted post-purification) forms, and is shown to be jointly regulated by the status of Fe-S clusters and Mg²âº. Typically ATPases require Mg²âº, as is true for PabABCE1, but Mg²âº also acts as a negative allosteric effector that modulates ATP affinity of PabABCE1. Physiological [Mg²âº] inhibits the PabABCE1 ATPase (K(i) of â¼1 µM) for both apo- and holo-PabABCE1. Comparative kinetic analysis of Mg²âº inhibition shows differences in degree of allosteric regulation between the apo- and holo-PabABCE1 where the apparent ATP K(m) of apo-PabABCE1 increases >30-fold from â¼30 µM to over 1 mM with M²âº. This effect would significantly convert the ATPase activity of PabABCE1 from being independent of cellular energy charge (φ) to being dependent on φ with cellular [Mg²âº]. These findings uncover intricate overlapping effects by both [Mg²âº] and the status of Fe-S clusters that regulate ABCE1's ATPase activity with implications to ribosomal function.
