Nucleotide Binding to Isolated Alpha and Beta Subunits of Proton Translocating Adenosine Triphosphatase Studied with Circular Dichroism

Abstract

The catalytic and allosteric sites of proton translocating adenosine triphosphatase (ATPase) were studied by measuring the binding of nucleotides to the ATPase, and its a and β subunits purified from thermophilic bacterium PS 3, with a circular dichroic spectrometer.
In contrast to mesophilic ATPases, this thermophilic enzyme contained no tightly bound nucleotides, and its subunits were stable after their purification. These properties were advantageous for analyzing both catalytic and allosteric sites. The former site showed rapid and loose binding, but the latter slow (t_1/2_??_1 h, for ADP) and tight binding.
When a nucleotide was bound, the g subunits showed a negative ellipticity at 275 nm corresponding to a tyrosyl residue, while the a subunits showed an ellipticity change corresponding to the absorption curve of the bound nucleotide. This difference enabled us to distinguish the binding sites in ATPase. At a low concentration, ADP selectively bound to a subunits in the ATPase, while at a high concentration, it bound to both subunits. This finding suggests that the tight binding sites are located in the α subunits. Although ADP and ATP bound to both the purified α and β subunits, CTP did not bind to β but only to α subunits, and ITP bound to β but hardly to α. These nucleotide specificities also supported the idea that the catalytic sites are located in the β subunits and the allosteric sites are located in the α subunits.