title: 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 alpha and beta subunits purified from thermophilic bacterium PS3, with a circular dichroic spectrometer. In contrast to mesophilic ATPases, this thermophilic enzmye 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 beta subunits showed a negative ellipticity at 275 nm corresponding to a tyrosyl residue, while the alpha 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 alpha 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 alpha subunits. Although ADP and ATP bound to both the purified alpha and beta subunits, CTP did not bind to beta but only to alpha subunits, and ITP bound to beta but hardly to alpha. These nucleotide specificities also supported the idea that the catalytic sites are located in the beta subunits and the allosteric sites are located in the alpha subunits.
mesh_terms: D000244:Adenosine Diphosphate; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate; D000495:Allosteric Site; D002942:Circular Dichroism; D046911:Macromolecular Substances; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D046911:Macromolecular Substances; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases